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TRANSACTIONS

OF THE

American Microscopical
Society

VOLUME XXXVII
1918

TRANSACTIONS

OF THE

American Microscopical
Society

ORGANIZED 1878 INCORPORATED 1891

PUBLISHED OQUARTEREY

BY THE SOCIETY

EDITED BY THE SECRETARY

VOLUME XXXVII

NUMBER ONE

Entered as Second-class Matter December 12, 1910, at the Post-office at Decatur,
Illinois, under act of March 3, 1879.

DEcATuR, ILL.
REvIEW Printinc & STATIONERY Co.

1918

TABLE OF CONTENTS

FOR VOLUME XXXVII, Number 1, January 1918

Insects as Carriers of Disease, by Malcolm Evan MacGregor............ Ps

Acanthocephala of North American Birds, with Plates I to V, by H. J.

Wianin Cleauerc cc rintras etl euler a heriocis ig a se oot ta scaMlaley s aleler ere) cya Sretteveh eet 19
Branchiobdellid Worms (Annelida) from Michigan Crawfishes, by Max
Wehbe Seg Set aa le ee MN ee 8 Se EA Leg trae rear 49

Notes and Reviews: A Chart on General Plant Histology and Physiology
(Plate VI), Raymond J. Pool; Method of Mounting Anatomical
Preparations for Exhibition, G. G. Scott; Green Light for Demon-
strating Living Cestode Ova, M. M. Ellis; A New Method of Stain-
ing Tissues Containing Nerves, Fontana’s Spirochete Stain, Simple
Method of Cleansing Old Slides, Menthol for Narcotizing, abstracted
by V. A. Latham; Freshwater Biology (Ward and Whipple); An
Introduction to the History of Science (Libby) ; A Short History of
Science (Sedgwick and Tyler); Biochemical Catalysts in Life and

Read easityy: CEE T ORIG ) shoe ots oh acd wah tence os ae ataecled ae ear at alan 4S Sane ane of fs 53
Minutes ot the Pittsbure Meeting. aie5 2.5 he soe aes ole aig Sas caeraeiaate 7i
PAAR SU ECCDOEE (6 a5, ois 8 oh altel visita kslosaiseoarehai ot Alga Ga monde sielkaitasene saree 72
WEES GEE SUMEDOEEN S: Go)) db nek snob etna son neh Who save EME, ks AS ate age 73

(This Number was issued on March 25, 1918)

OFFICERS

President =) No. ES. (GRIRVING 2 ois. 52! 40,5 Sis cies s amie bole eee sires Pittsburg, Pa.
First Vice President: H.M. WHELPLEY................-0000- St. Louis, Mo.
Secona Vice President: (GC. (0. ESTERLY ..oe sos 33 ove sie ne Los Angeles, Cal.
Secretar ye) (che WoGAEEOWy os ade. Ssidieid Selete weal ete bere ole wie eee one Beloit, Wis.
Dv CRSUFEKS ERS yc VAM OCTBAVE! « S'c'5.8 2 eis Sinks oe Da Ale o oe aie Urbana, IIl.
Gustodian> | MAGNUS (EFEAUME Halas Siete. soviet ee lantels kore se iets Meadville, Pa.

ELECTIVE MEMBERS OF THE EXECUTIVE COMMITTEE

1 ey INTE Dh ee a ee ASS AEG "2 3 Boulder, Colo.
DESPRE RERT Soe aye airs wie ais abla cee 5 50 p siaieis wi a ele Svs sib lelesopginibl arn Sine Manhattan, Kas.

EX-OFFICIO MEMBERS OF THE EXECUTIVE COMMITTEE
Past Presidents Still Retaining Membership in Society

ALBERT McCa.ta, Ph.D., F.R.M.S., of Chicago, Ili.
Geo. E Fett, M.D., F.R.M.S., of Buffalo, N. Y.,
Simon Henry Gace, B.S., of Ithaca, N. Y.,

at Chicago, IIl., 1883
at Detroit, Mich., 1890

at Ithaca, N. Y., 1895 and 1906

A. Currrorp Mercer, M.D., F.R.M.S., of Syracuse, N. Y.,
at Pittsburg, Pa., 1896

at New York City, 1900

at Denver, Colo., 1901

at Winona Lake, Ind., 1903
at Sandusky, Ohio, 1905

A. M. Briere, M.D., of Columbus, Ohio,

C. H. E1icenmann, Ph.D., of Bloomington, Ind.,
E. A. Birce, LL.D., of Madison, Wis.,

Henry B. Warp, A.M., Ph.D., of Urbana, IIl.,

S. lumb hio, 4 (
Herpert Oszorn, M.S., of Columbus, Ohio ai Minacapalie ans gid

at Washington, D. C., 1911
at Cleveland, Ohio, 1912
at Philadelphia, Pa., 1914
at Columbus, Ohio, 1915
at Pittsburg, Pa., 1917

A. E. Hertzver, M.D., of Kansas City, Mo.,

F. D. Heap, Ph.D., of Philadelphia, Pa.,
CHARLES BrooKover, Ph.D., of Louisville, Ky.,
Cuartes A. Kororp, Ph.D., of Berkeley, Calif.,
M. F. Guyer, Ph.D., of Madison, Wis.,

The Society does not hold itself responsible for the opinions expressed
by members in its published Transactions unless endorsed by special vote.

TRANSACTIONS

OF

American Microscopical Society

(Published in Quarterly Installments)

Vol. XXXVII JANUARY 1918 No. 1

INSECTS AS CARRIERS OF DISEASE
By Matcotm Evan MacGrecor
Wellcome Bureau of Scientific Research

So much has been said about insects since the War began that
it is, I think, advisable that some attempt should be made to sum-
marize our knowledge of the more important insect-borne diseases
and their vectors. While insects have long been suspected of being
responsible for the transmission of serious diseases, it may be said
that practically the whole of our knowledge of insects in this rdle
has been acquired within the past twenty years. So rapidly, how-
ever, has the charge of this offence been made out against them
that, although it is common knowledge they have been proved guilty,
it is not generally realized upon how many counts the verdict rests.

It has lately been my good fortune to give class instruction
for the War Office to officers of the R. A. M. C. who are proceed-
ing to the East, and, in order to bring home to my audience the
importance of the connection between insects and disease, I have
compiled the tables which I now publish. These can in no way
claim to be complete, but merely present the more important insect-
borne diseases, including important human diseases that on certain
grounds are suspected of having insect vectors. With these tables
I also publish one (Table VI) which includes the chief insects and
acarina that are directly the cause of disease in man and his domestic
animals. To complete the list of insect-transmitted diseases would
demand the consideration not only of other mammals as hosts,
but also of avian and reptilian hosts. In the present instance this
would be to carry the subject beyond general interest, but it must be
remembered, therefore, that, long as the present list of charges
is, insects are not here arraigned on all the counts that might with
justice be preferred against them.

Reprinted from Journal of Tropical Medicine and Hygiene, Sept. 15, 1917; xx, 18.

8 M. E. MAC GREGOR

During the last few years medical entomology has been rapidly
establishing itself an an invaluable branch of preventive medicine,
and with the outbreak of the present War a great deal of interest
and study has been devoted to this subject in Europe, notably in
connection with the transmission of typhus fever by lice, and the
dissemination of bacteria and other organisms by flies. Moreover,
the importance of insect vectors has been generally realized, and
many of the astonishing interactions between pathogenic micro-
organisms and certain arthropoda have become popular knowledge.

Centuries ago insects were suggested as being possibly con-
cerned in the spread of disease, and from time to time such logical
hypotheses were advanced that it is surprising that the establishment
of the truth was not sooner forthcoming. In 1577 Mercurialis, an
Italian physician, suggested that plague, which was then ravaging
Europe, was spread by flies feeding upon the diseased and dead, and
later depositing feecal matter on food consumed by healthy persons.
This idea held ground, and various suggestions occur as to the
spread of disease by flies in the literature of the eighteenth century.
Edward Bancroft in 1769 advanced the theory that “yaws’” was
transmitted by flies feeding on diseased subjects, and carrying the
contagion by settling on open wounds or scratches on healthy per-
sons.

In 1848 Dr. Josiah Nott, of Mobile, Alabama, published a
remarkable article in which he gave reasons for supposing that
yellow fever was an insect-borne disease. However, although he
mentioned many insects, he did not specify any insect as the particu-
lar vector.

The connection between malaria and the mosquito had long
been held, it is said, by the Italian and Tyrolese peasants, and even
by the natives of East Africa, but the first charge brought against
the mosquito in the spread of disease by scientific authority was in
connection with yellow fever.

In 1853 Dr. Daniel Beauperthuy, a French physician, wrote
ably arguing that yellow fever and other fevers were transmitted by
mosquitoes, but in those days there being no accepted belief in a
contagium vivum, he concluded that the virus was obtained from
decomposing material that the mosquito had consumed, and which

INSECT CARRIERS OF DISEASE )

was later accidentally inoculated into man. MRaimbert in 1869
showed by experiment that anthrax could be disseminated by flies.

Epoch-making in the history of our knowledge of insect vectors
was Manson’s discovery in 1878 that Filaria bancrofti was spread
by mosquitoes, but at first he thought the filarie escaped from the
insect into water, and reached man in this manner. Later work
by Manson and his colleagues determined the exact means of trans-
mission.

It was not until twenty-eight years after Beauperthuy’s theory
that Charles Finlay, an American of Havana, in 1881 definitely
attributed the transmission of yellow fever to a mosquito of a
definite species. He had noticed the connection that seemed to exist
between the presence of large numbers of Stegomyia fasciata and
the prevalence of yellow fever. He then attempted to transmit
the disease experimentally by the bites of this mosquito, and al-
though his experiments are open to criticism, there is no doubt that
he did succeed in doing so.

Three years later, in 1883, another American, A. F. A. King,
advanced the first well formulated mosquito-malaria theory, and in
1898 Ross, in India, demonstrated beyond doubt the important role
played by mosquitoes in the transmission of malaria.

In 1899 the American Yellow Fever Commission (Reed, Car-
roll, Lezear, and Agramonte) were sent to Cuba, and were there
able to demonstrate with certainty that yellow fever is transmitted
by S. fasciata.

It is interesting thus to note the almost parallel development
in time of our knowledge of two of the most important insect-borne
diseases. To deal even briefly with the historical aspects of our
knowledge of other diseases tabulated below would be to consume
a large amount of space, and the foregoing account will have indi-
cated the path that has led to subsequent discoveries whose histories
are readily available.

I will pass, therefore, to a few notes on each of the tables.

Notes To TABLE I.—DIsEASES OF UNKNOWN ORIGIN
The majority of these diseases are doubtless caused by living
viruses; often organisms of ultra-microscopic size, and commonly
referred to as “filterable viruses.”

10 M. E. MAC GREGOR

In the case of pellagra it would appear, however, from the
most recent work that, although it is still considered by many
persons to be a possible insect-borne disease (and, according to
Sambon, having a likely vector in either the Ceratopogonine or
Simulude), Goldberger in America considers it a disease now cer-
tainly attributable to vitamine-starvation through an wnbalanced
diet. If this is the case, there is no causative organism and no vector,
and pellagra should be ruled out of present consideration. The
question, nevertheless, is by no means settled.

The virus of acute anterior poliomyelitis is still not isolated
with certainty. Flexner and his colleagues have been able to culti-
vate a filterable micro-organism which produced the disease in
experimental animals, and more recently Rosenow and his fellow
workers have isolated a polymorphous streptococcus, with which
they were also able to proauce paralysis in certain animals. Nuzum
and Herzog were able to do likewise by a Gram-positive micrococcus
isolated from the brain and spinal cord of persons dead from the
disease.

Poliomyelitis has been very generally suspected of being trans-
mitted by insects, particularly by Stomoxys calcitrans (the stable-
fly), fleas, and Tabanide (gad-flies). Nevertheless, it appears more
likely that it has an eerial transmission, infection being acquired
through the buccal and nasal mucous membranes.

The causative organism of Rocky Mountain spotted fever, Wol-
bach claims to have discovered in the bodies of infective ticks (Der-
macentor venustus ).

Notes To TABLE JI.—DIsEASES OF BACTERIAL ORIGIN

In the majority of cases, diseases of this class have an indirect
transmission by insects—that is to say, instead of the organism
entering the body of the host through inoculation by the bite of an
insect (direct transmission), the organisms are carried in or on the
insect’s body, and are deposited by contact on human food or skin
abrasions, and in this manner cause infection.

Bacillus tuberculosis may be disseminated by house-flies feeding
on infective sputum, as was first shown by Spillman and Haushalter
(1887), and subsequently by the investigations of other workers.

N. B.—Names between

Tasce I,

THE MORE IMPORTANT INSECT-BORNE DISEASE OF UNKNOWN ORIGIN

square brackets = certain vectors; names without square brackets = probable vectors; names followed by ? = possible

vectors.

Organism Host Disease Vector

De reigre fren ro ean aot Dengue (breakbone fever) er [Sandflies (Phlebotomus), Mosquitoes. C. fatigans; S. fas-
Three-day fever, syns. “Dog ciata] ;
Poseurs iets Hare Me ates disease,’ Sandfly fever, Phle- {Sandflies (Phlebotomus), Mosquitoes. C. fatigans; S. fas-
botomus fever ciata]
Ceeraieve etaie cele de AO Yellow fever noe ee [Mosquitoes (Stegomyia fasciata)]
Ree stete ote are we 500 Trench fever SOE rays Lice ?
? Salivary toxin? she dy oBe Tick paralysis (American) ciate [Ticks, (Dermacentor venustus)]
? Salivary toxin? fre ee wae Tick paralysis (Australian) est [Ticks (Ixodes ricinus)]
Papestars atio eee 2 Be Rocky Mountain spotted feve [Ticks (Dermacentor venustus)] :
Petetere teat ate Ud Ae Japenese river fever (shima [Mites (Larval trombidiide)] “aka mushi’”
mushi
Reetatas ware SAG bu ae Acute anterior poliomyelitis siene ? Many insects have been claimed as vectors, notably
Stomoxys calcitrans?
Pees mate ees dg wie Pellagra Save tan acre ? Gnats of the genus Simulium have been claimed?
Poeiais inv eee ? sats Typhus fever site eee [Lice]
- Taare II.
THE MORE IMPORTANT INSECT-BORNE DISEASES OF BACTERIAL ORIGIN
N. B.—Names between square brackets = certain vectors; names without square brackets = probable vectors; names followed by ? = possible
vectors.
The word “flies” includes in the main: Musca domestica, Fannia sps., Calliphora sps., Lucilia sps., and Sarcophaga sps.
Organism Host Disease Vector

Bacillus anthracis els iets .».» Man and animals Anthrax aye ... Flies], Tabanid@? Beetles? F

zy dysenterie ... thes wie’ ot: ere --- Bacillary dysentery dite [Flies] (Musca domestica, Calliphora sps.,

Lucilla _sps.) :
zh lepre aiale eine tele id 400 -.. Leprosy wee ahi LIES Fleas? Bed-bugs? Skin mites?
: : Mosquitoes?

# paratyphosus A arene wane ae ac ae Paratyphoid fever ats [Flies]

a B ofthe “aes ide Fate aes 4 # Ee a eesl

2 pestis a tere arts dire ” and rats ... Plague ACD ..-. [Fleas]

4 tuberculosis aiie aCe ” and animals Tuberculosis ... ... Flies, cockroaches, fleas? Bed-bugs?
Bacillus typhosus Riise ae verste wee Lae pense Dy DHOIantever -»- [Flies]
Bartonia bacilliformis «x-bodies stake ie fee cee Verruga AG ... [Phlebotomus verrucum] :
Spirillum cholere SAD wile ave dd shale -.-- Cholera arts ... [Flies], cockroaches, ants. Although the main

channel of infection is the consumption of
infected food and water

12 M. E. MAC GREGOR

With the high vitality and resistance to drying possessed by the
B. tuberculosis, the possibly long incubation period within the body
and the insidious onset of the disease, the danger from Musca
domestica in this connection is still not sufficiently recognized.

Human infection with plague and typhus has been shown to
be acquired principally by the entrance of the virus through skin
lesions, the insect vector having been crushed either during or
after the act of blood-sucking. The stomach contents or infected
excreta may be rubbed into the lesions or gain entry through
abrasions caused by scratching.

This, however, does not preclude the possibility of direct infec-
tion also occurring, at least sometimes in the case of plague, as the
infected flea has the proventriculus occluded by the plague organ-
isms when the flea infection is at its height. Septiceemia following
mosquito bites occasionally happens, and as likely as not the path-
ogenic organisms are introduced when the mosquito bites. Direct
transmission by blood-sucking insects may possibly also occur in
certain instances in the spread of tuberculosis and leprosy.

If Wolbach’s organism (see Table I and notes thereto) is
proved to be the cause of Rocky Mountain spotted fever, this will
also be a disease of bacterial origin with direct transmission through
a tick, Dermacentor venustus.

Notes To TABLE II]I.—DIsEASES OF SPIROCHZTAL ORIGIN

With these diseases the usual method of transmission is direct
—that is to say, through the bites of the insect vectors.

Exceptions occur in the case of relapsing fever transmitted
indirectly by lice, and yaws where Musca domestica may at times
convey the organism from diseased to healthy persons.

Notes To TABLE IV.—DISEASES OF PROTOZOAL ORIGIN

Both direct and indirect methods of transmission by insects
occur with diseases of this class. With the intestinal parasites,
indirect transmission takes place by the flies feeding on feces con-
taining the resistant stages (cysts), and later depositing them on
human food and drinking water either by regurgitation of the
stomach contents, or more often per anum, as Wenyon and O’Con-

c

Organism Host

Micrococcus melitensts ead Ane

Diplococcus intracellularis —... on Man
fs pemphigi contagiost mies yu.

Taste IJ.—(Continued.)

Tropical impetigo

Tasle III.

Disease
Man and goats... Undulant fever; syns.
Malta fever, Mediter-
ranean fever, Remit-
tent fever
aire ... Cerebrospinal fever ...

Vector

[Flies]. Although the main channel of infec-
tion is the consumption of goat’s milk

Flies?

[Lice]

THE MORE IMPORTANT INSECT-BORNE DISEASE OF SPIROCHATAL ORIGIN

N. B.—Names between square brackets = certain vectors; names without square brackets = probable vectors; names followed by ? = possible

vectors.

The word “flies” includes in the main:—Musca domestica, Fannia sps., Calliphora sps., Lucilia sps., and Sarcophaga sps.

Organism Host
Spirocha@ta carteri ROrhS Soe Man Aen
nde duttont Rec ort " erage
a! gallinarum ateceomeentey Fowls Aa
4 novyi Siraawe tele Man wits
zy pertenuis Seren WY apc
” recurrentis AC eerie, Lu cee
a berbera Steen te aie

Disease

Indian relapsing fever
African 2
Spirochetosis ai
American relapsing fever ...
Yaws (Frambeesia) aie
European relapsing fever ...
North African relapsing fever

.

TaBLeE IV.

eee

Mel (Tick fever) Wes

Vector

[Lice]

[Ticks (O. moubata, O. savigny))
[Argas persicus]

[Lice]
Flies?
[Lice].
[Lice]

Bed-bugs?

THE MORE IMPORTANT INSECT-BORNE DisEAsES OF ProTozoAL ORIGIN

N. B.—Names between square brackets = certain vectors; names without square brackets = probable vectors; names followed by ? = possible

The word “flies” includes in the main:

Organism Host

Entameba histolytica -.. Man ares
Lamblia intestinalis* Pare 3 seme
Plasmodium malarie sere As Noo
4 vivax ae 2 arate

ay falciparum oa =F are

vectors,
Disease
... Ameebic dysentery
... Flagellate ” ere

«+» Quartan malaria ...—
... Benign tertian malaria
... Malignant or subtertian

malaria

Musca domestica, Fannia sps., Calliphora sps., Lucilia sps., and Sarcophaga sps.

Vector
... [Flies]
... L[Flies)
..- [Anopheline mosquitoes]
... [Anopheline mosquitoes]
... [Anopheline mosquitoes]

14 M. E. MAC GREGOR

nor have shown recently.t_ Needless to say, infection also occurs—
and perhaps principally—by mechanical and erial transmission of
the cysts to food and water.

The majority of the protozoal blood parasites have insect
vectors, on which they depend solely for transmission, and in cer-
tain cases these vectors are specific: Malaria, Anophelines; sleep-
ing sickness, Glossine ; European relapsing fever, Pediculi. Other
insect-borne blood parasites are apparently able to be transmitted by
more than one vector, i. e., Kala-azar, bed-bug (Patton) ; kala-azar,
Triatoma rubofasciatus (Donovan); Souma (Trypanosomiasis),
Glossine ; Souma (Stomoxys calcitrans).

Notes To TABLE V.—DISEASES OF HELMINTHAL ORIGIN

With the exception of possible infection with certain helminths,
resulting from the carriage by flies of helminth ova from feces
and subsequent deposition of the ova on food, the insect-borne
helminths all undergo part of their life-history in the body of the
insect vector. Thus the adult Filaria bancrofti live in human
lymphatic glands. The ova find their way into the blood-stream,
where they hatch to the Microfilariz, and some are taken up from
the blood when a mosquito bites a person harbouring the organisms.
These, if they have entered the stomach of Culex fatigans, or other
intermediate host, soon make their way to the thoracic muscles of
the mosquito, where they undergo definite metamorphosis. When
this is complete (usually in from sixteen to twenty days) the worms
make their way into the mosquito’s proboscis, and when next it
pierces the skin of some victim the filariz burst through the pro-
boscis sheath and make their own passage through the skin, from
which they soon travel to some lymphatic gland, where they become
sexually mature, and the cycle is repeated. Similarly, Dipylidium
caninum passes part of its life-history in the rat flea, and becomes
sexually mature in the dog or man. The ova are ingested by the
larval flea, and infection by the cysticercoid stage follows the acci-
dental ingestion of the flea by the definite host.

“The Carriage of Cysts of Entameba histolytica and other protozoa, and eggs of
Parasitic Worms by House-flies, with some Notes on the Resistance of Cysts to Disinfect-

ants and other Agents.” C. M. Wenyon and F. W. O’Connor, Journal of the Royal Army
Medical Corps, May, 1917, p. 522.

TasLe IV.—(Continued.)

Organism Host Disease Vector
Leishmania tropica eee . ae ... Oriental sore re: eat -». Flies? Fleas? Phlebotomus?
Hippobosca?
ee donovani fev Le stare ... Kala-azar Ath Sere ... Bed-bugs? Fleas? Triatoma?
tt sp. incerta naw ste Mere ... Espundia Re ave ..» Probably some tropical blood-sucking
Insect
2 infantum ... Children Nat ... Leishmaniasis aL Rare ..+ Fleas?
Trypanosoma gambiense -». Man aves ..- Sleeping sickness... RG ... [Glossina palpalis (Tsetse flies)]
ae rhodesiense eine 4 moe pare | Leon aad ... (Glossina morsitans) ” u
2 brucei ... Cattle and horses ... Fly sickness (Nagana) ies ... (Glossina morsitans) _” Ps
td lewisi en ate Grave ... Rat trypanosomiasis aleve ... Rat louse? and [Rat fleas (Ceratophyllus
fasciatus), Ctenocephalus canis]
bg evansi .-.- Horses, mules, camels Sutras acs core aie tere io flies (Tabanidew), Tabanus striat-
us
Schizotrypanum crusi --» Man AP --» Chaga’s disease ... sera ... [Triatoma (Conorhinus) megistus]
Babesia bigeminum eee Cattle stale -.. Red-water fever ... nae ... [Ticks (Margaropus annulatus))
td ovis ane «++ Sheep aac ..- Piroplasmosis Seto Ante ..» [Ticks (Rhipicephalus bursa)]
fe! canis mais wre WORE sere ... Malignant jaundice ee ... [Ticks (Rhipicephalus sanguineus and
Hemaphysalis leachi)]
” caballi Bite ... Horses and mules ... Piroplasmosis tiare On ... [Ticks (Dermacentor reticulatus)]
Nuttallia equi Ge cere Us ” atin ot Rae ara ... (Ticks (Rhipicephalus evertsi)]
? Chlamydozoa ? atic Perey Evel AGO .»» Ophthalmia egyptica and other ophthal- [Flies]

mic conditions
*By some authorities Lamblia intestinalis is not regarded as pathogenic,

TaBLe V.
THE MORE IMPORTANT INSECT-BORNE DisEASES OF HELMINTH ORIGIN

N. B.—Names between square brackets = certain vectors.
The word “flies” includes in the main: Musca domestica, Fannia sps., Calliphora sps., Lucilia sps., and Sarcophaga sps.

Organism Host Disease Vector
Dipylidium caninum ... Man and dogs ... Teniasis (tapeworm) ... ... [Dog louse (Trichodectes latus)]; [Dog flea
(Ctenocephalus canis)]; and the [Human flea
(Pulex irritans)]

Ova of certain helminths Man Ati ... Helminthiasis (parasitic worms) [Flies] |

Microfilaria bancrofti ite « at ... Filariasis (Elephantiasis) ... [Mosquitoes (Culex and Anopheles sps.)]

Loa loa trae in Ms ScrC .». Calabar swellings aoe Ah Hees fiies (Chrysops dimidiata and Chrysops
silacea

Filaria immitis. | .». Dogs ie ... Dog filariasis are ... (Mosquitoes (Culex and Anopheles sp.)]

Hymenolepis diminuta Rats, and occasionally Tzniasis ce aes no Lees)

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INSECT CARRIERS OF DISEASE 17

Notes To TABLE VI.—Dtseases DiREcTLY ATTRIBUTABLE TO
INSECTS AND ACARINA

In each case the disease results from the damage done by the
insects and acarina in adopting existence upon the body of the
host and living upon its tissues. The time spent upon the host
may cover the whole life-history of the parasite and many subse-
quent generations, as with Sarcoptes scabiei and the Pediculi, or it
may only embrace part of the parasite’s development, as with the
larve of flies causing myiasis. In either case the injury to the host
may be so extensive as to cause death from the loss of vital tissue,
or the injury itself, although insignificant, may indirectly cause
death to the host by providing a suitable path for invasion by patho-
genic micro-organisms.

CONCLUSION

It should be borne in mind that a large number of diseases
included in the foregoing tables are not confined to being spread by
insects, and insect transmission may in some cases only be occasional.
This fact, however, cannot afford the preclusion of such diseases
from consideration, and where transmission of the indirect type is
possible, it is obvious that we are unable to form any just estimate
of its relative importance. Probably, nevertheless, the dissemina-
tion of pathogenic micro-organisms by flies, for all that has lately
been said in this connection, has not even yet been over-emphasized.
It seems more than likely that Bacillus tuberculosis is spread in this
manner from infective sputum to food (milk particularly) to a
much greater extent than is commonly imagined, and there being
no probability of rapid acute infection, as with B. typhosus, the
part played by the fly is too apt to be overlooked.

Much of our knowledge with regard to insects and disease
is still indefinite, as may be seen from the tables, but to anyone
not particularly conversant with the subject, what we already know
with certainty, even in connection with only the more important
diseases that have been considered, may be sufficient to cause
some little surprise.

THE ACANTHOCEPHALA OF NORTH AMERICAN BIRDS*

VIII.

H. J. Van CLEAVE
CONTENTS

AER OCUIETECIEN IM oh eens 2 ane kaa fed oe otto al fave Reta Urea
ene) Gents) COtyneSOmia.: ois Jaze aye <5 6 odele!-\u rd os Sal piaecal sto lesa
Corynosoma constrictum NOV. SPEC.... 6... cece cere eee e eee eee
(he Genus PlaciothyncHus.. oi. is)i5e alas odes «lec elemee tees sis
Plagiorhynchus rectus (Linton) ..........00cceeseesereeeeee
Plagiorhynchus formosus nov. SP€C...... 0+. ee eee cece ee eeees
hie Gebus \Polymorphes: <<) 5:6)... s\< se-6 6 olsieeretele,sineles vale cwieis
Polymorphus obtusus NOv. SPCC.... 2... cece cece eee eee e renee
LUD GLANS, SOE Oe opis ct le oie vias pie ele: ci biota wisialvisiai eal ore lei oisin)» os /4)5 si
Mie GedusTOentrornyHCHUsy cy vidas sie bac ele ceiw'ereln nae a eer
he Genus Mediorhiynchus’ |... <jjs:e/seice's slecis eo siaerina aime
PPHE GETS TICEELODIIS) | occ cere dials fee chers 4 cic cto alates, elesel esa sialaleia’e
Heteroplus grandis (Van CC.) ........ccccccceeceseesccrcons
MURe WMG emiay HALLICOMIS® (ro ctascise'cieers, a2 vein sible oh die aie </alalemreiatetale Hae
ite Genus Arhythmorhynchus. . (46). cj5 2 \0\s/< a oe ite a caciemiatale
Species Inquirendze and Species of Doubted Determination. .
fEchinorhynchus pict collaris Leidy .............eeeeeeee
?Echinorhynchus caudatus Zeder of Leidy ...........+....
?fEchinorhynchus striatus Goeze of Leidy .............+4..
?Echinorhynchus hystrix Bremser of Leidy................-
Distribution of Acanthocephala of Birds.................---
Occurrence of Genera of Acanthocephala in Families of Eu-

ropean and North American Birds... 00.00.66 056 0000+
Genera of Acanthocephala with the Orders of Birds from

CUT CHIN RECOLGEGIN se clera eee elo Nataberniae teeta ty CTC EN sta Nenel alti ste ail
Rey ito the Genera and Species) 1/2 /).4c-itsewisaiee nae ele t.tiercyalsiains
Seta Vs et tne eter leta rs elias coacarer suis trie cerate ra sieeal ehetausiete enero ate laelaleye
MNECEAPUEG CALC ois g ciarcialtieraid ate ee 4 SIE A ea ARS olathe A: wae
ReMANO OLE NALES 2 0c 2 ae cual ave ele wlels. shel eue/wleversib vain ite s,8) ele.

I. INTRODUCTION

Parasitologists have given little attention to the Acanthocephala

parasitic in North American birds.

The result has been a rather

general belief that infestation with Acanthocephala in this class of

vertebrates is rare.

It is true that heavy infestations are more

commonly found in other groups of vertebrates, but many of the

*Contributions from the Zoological Laboratory of the University of Illinois, No. 104.

20 H. J. VAN CLEAVE

water and shore birds carry very heavy infestations of Acanthoce-
phala. Among the land birds, while infestation is not rare, it is
usually of less frequent occurrence, and the number of parasites
found in a single host individual is usually smaller. Most of the
earlier records of the occurrence of Acanthocephala in North Ameri-
can birds ascribe the forms found to known European species. This
is not surprising when one reviews the extent to which the study of
Acanthocephala in birds has proceeded in Europe. Yet the mere
fact that all earlier writers recognized but the one genus, Echino-
rhynchus, in part explains their lack of appreciation of specific char-
acters within this group. On the whole, most of the earlier specific
descriptions are little more than sufficient to permit of later workers
recognizing the genus to which the species belong. This lack in
specificity of earlier descriptions quite naturally lead the new world
investigators to believe that the species they found on this continent
were the same as the European species, since the descriptions pub-
lished contained insufficient data to permit of a separation.

As indicated by the writer in an earlier paper (Van Cleave
1916 b: 228), the acanthocephalan fauna of North America is in
the main a distinctive fauna with few species identical with those
of the European fauna. The attempt on the part of earlier para-
sitologists to ascribe names of European species to forms found
on this continent has, to a great extent, hindered the appreciation
of this distinctness of the North American fauna, and at the same
time has lead to considerable confusion regarding the geographical
distribution of the genera and species of Acanthocephala. Unfor-
tunately, many of the specimens of the older writers are not avail-
able for restudy to determine the correctness of the original deter-
minations. However, in most cases where further study has been
possible points of difference from the European species have been
found too numerous to permit of including the American forms in
the European species.

The writer has made an extensive study of the Acanthocephala
of birds in which he has had access to the collections of the U. S.
Bureau of Animal Industry, the U. S. National Museum, the Marine
Hospital Service, private collections of Dr. H. B. Ward and of the

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS Z1

writer. Papers covering the species of several genera of Avian
Acanthocephala have already been published by the writer as a
result of the study of these collections. The present article deals
with the descriptions of several new species and a reconsideration
of some forms previously described, but here for the first time
ascribed to the proper genera as recognized by more recent develop-
ments of classification of the Acanthocephala. In all cases the study
has been made from cleared, stained specimens mounted in damar.

Linton (1892: 92) recorded the occurrence of Echinorhynchus
striatus Goeze from the intestine of the black scoter, Oidemia ameri-
cana, collected at Yellowstone Lake, Wyoming. The writer has
reexamined the original material of this collection and finds that
these individuals constitute an undescribed species of the genus
Corynosoma. Linton (page 92) remarked that the two females
constituting the extent of the infestation of one of the hosts differed
from the six males from the other host individual, in the “absence
of spines at the posterior end” of the former. It is true that this
sexual dimorphism is characteristic of the members of the genus
Corynosoma as indicated by the founder of the genus, Luhe (1904
and 1911).

Of the materials from this collection deposited in the U. S.
National Museum, one entire female, a portion of a second and
five of the males, have been studied by the present writer. The
entire female specimen is immature but, fortunately, the fragment
of a specimen is of the anterior end of a fully mature individual
so that the body cavity contains well developed embryos.

II. THe Genus Corynosoma Lihe, 1904

The genus Corynosoma was established by Lithe to accommo-
date two species of Acanthocephala parasitic in fish-eating mammals
and birds. This genus is characterized by the presence of spines
on the anterior end of both sexes while in addition the males bear
spines around the genital opening. The body is swollen anteriorly
and with its coating of spines adheres to the intestinal mucosa of
the host thereby furnishing an accessory means of attachment. Pos-
teriorly the body gradually becomes smaller.

ZZ H. J. VAN CLEAVE

Corynosoma constrictum nov. spec.
Plate I, Figures 1, 2 and 3
Synonym: Echinorhynchus striatus Goeze of Linton 1892.

SPECIFIC DEFINITION. With the characters of the genus. Body
of the males 2.28 mm. to 4.3 mm. long, with a maximum diam-
eter of from 0.5 mm. to 0.6 mm. Linton gives the measurement
of a female 3.3 mm. long with a diameter of 0.8 mm. Proboscis
slightly larger at base than at tip, armed with sixteen longitudinal
rows of ten to twelve hooks each. Hooks near base of proboscis
0.035 to 0.041 mm. long, near middle of proboscis 0.041 to 0.047
mm. long, near tip of proboscis 0.030 to 0.041 mm. long. A con-
striction occurs around the body at about the anterior third (see
Fig. 1). In both sexes the part of the body wall anterior to this
constriction is armed with small cuticular spines about 0.030 mm.
long. Each of these spines is embedded in a triangular elevation
of the cuticula projecting from the general body surface. In the
males there occurs in addition a group of cuticular spines surround-
ing the genital opening (Fig. 2). The genital spines are of the
same size as those on the anterior region of the body, usually with
the tip strongly recurved. Embryos in body of female 80 to 108
long by 12 to 16n wide (Fig. 3).

Type host Oidemia americana, in intestine. Type locality Yel-
lowstone Lake, Wyoming. The cotypes of this species are deposited
in the Smithsonian Institution ; the males under catalog number 5449
and females under number 5439.

III. Tuer Genus PLAGiorHyNcHUS Lithe, 1911

In the same paper mentioned above (1892: 91) Linton described
another species of Acanthocephala from the intestine of a gull taken
at Guaymas, Mexico. To this new species, founded on the study
of one male and one female, he gave the name Echinorhynchus
rectus. The present writer has examined the female of this col-
lection, the only specimen in the bottle of material turned over to
him for study from the collections of the U. S. National Museum.
Facts brought out by the reexamination of the female of this species
and the study of Linton’s description of the male and female clearly
indicate that this species belongs in the genus Plagiorhynchus, which

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 23

up to the present time has not been recorded as occurring in North
America. In the present paper the writer describes still another
species belonging to the genus Plagiorhynchus.

CHARACTERS OF THE GENUS PLAGIORHYNCHUS. Acanthocephala
belonging to this genus are parasitic as adults in the alimentary
canal of birds. The proboscis is cylindrical with numerous hooks
arranged in radial symmetry. The body proper, which is entirely
devoid of spines, is usually short, elliptical, or with a tendency
toward egg-shaped. The proboscis receptacle is a double walled
muscular sac attached at the base of the proboscis. In dealing with
other genera of Acanthocephala the writer has found that in Arhy-
thmorhynchus (Van Cleave 1916:a) the embryos in recently discov-
ered American species differ in appearance from those described for
European species of obviously the same genus. Here again in the
genus Plagiorhynchus Lihe (1911:27) described embryos and fig-
ured one having the middle membrane with a conspicuous spherical
knob at each of its two poles. This he considered characteris-
tic of the entire genus (1911:26). The embryos of P. formosus
(Fig. 6) are elliptical with no polar knobs. Here is evidence, in
addition to that previously brought out by the writer (1916:a) and
mentioned above, regarding the advisability of omitting the shape
of the embryonic shells from the generic diagnosis.

Luhe (1911:26) specified the presence of six closely com-
pacted, thickset, cement glands as characteristic of the genus Plagi-
orhynchus. In earlier papers (1914 and 1916) the present writer has
shown that shape and number of cement glands may both vary
widely among different species of the same genus. Consequently
I propose that a fixed number of cement glands and the shape of
the cement glands be omitted from the list of characters diagnostic
of this genus. Descriptions of two species, one new, and the other
newly attributed to this genus follow.

Plagiorhynchus rectus (Linton, 1892)
Plate I, Figure 7
Synonym, Echinorhynchus rectus Linton, 1892

Described originally from one male and one female ef which
the female only has been reexamined by the present writer. Body

24 H. J. VAN CLEAVE

of female 9 mm. long and 0.8 mm. in diameter. Proboscis cylin-
drical, 19 mm. long; 0.26 mm. in diameter; armed with twenty-
four longitudinal rows of about twenty hooks each. In specimen
examined this last number was calculated on the basis of the num-
ber of hooks on the exposed portion of the proboscis, since the tip
of the proboscis is inverted. Hooks near the base of the proboscis
0.082 mm. long, with a diameter of 0.016 mm. at the point of
emergence from proboscis wall; hooks near middle of proboscis
0.070 mm. long, recurved, with a diameter of 0.020 mm. at the
point where the hooks curve backward; hooks near tip of pro-
boscis 0.053 mm. long. Male 8.8 mm. long and 0.8 mm. in diameter.
Testes oval, approximate, median. Female not fully mature so
measurements of embryos cannot be given.

Type host, Larus (Chroicocephalus) sp. taken at Guaymas,
Mexico. Type female deposited in U. S. National Museum, cata-
log number 5431.

Plagiorhynchus formosus nov. spec.
Plate I, Figures 4, 5 and 6

SPECIFIC DEFINITION. With the characters of the genus. Body
about 10 mm. long, elliptical to slightly ovoid. Proboscis practically
cylindrical, diameter about one-third of length; armed with sixteen
longitudinal rows of thirteen to fourteen hooks each. Cement glands
long, tubular. Hard shelled embryos inside body of female ellip-
tical, 48 to 60u long by 12 to 20u in diameter.

This species is described from four mature individuals, two
males and two females, in the Parasite Collection of the U. S. Bu-
reau of Animal Industry; catalog number 4598. The writer desig-
nates one male and one female as types of the species. Descriptions
of these follow.

TYPE MALE. Body elliptical with anterior and posterior ex-
tremities slightly flexed ventrally; entire length 8.5 mm.; maximum
diameter 2 mm. Body proper devoid of spines. Proboscis sub-
cylindrical, length 1.06 mm., diameter 0.33 mm., armature as men-
tioned in definition of species. The following table of measure-
ments of hooks in a single row from tip to base of proboscis
indicates relative size of hooks in various regions of proboscis.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 25

RET Wa Io 0)c 3ieher'se/'at «16 Tens) Al SRG) Se oS MR BM | EZ ES
Length in w...... Fi FF BS) (8S) SI SSN BS 83): SS Fao), 77), Oo) ease

Proboscis receptacle cylindrical, 1.73 mm. long, 0.42 mm. in
diameter, base of the receptacle rounded, with invertors of pro-
boscis penetrating posterior extremity (Fig. 4). Brain 0.180 mm.
long, in center of proboscis receptacle, between the invertors. Retin-
acula conspicuous at the middle of the proboscis receptacle. Lem-
nisci 0.192 mm. long, 0.058 mm. in diameter. Anterior and posterior
testes form an oblique line of contact; each 1.15 mm. long and 0.6
mm. in diameter. Cement glands long, tubular, extending from the
dorsal margin of posterior testis to region of bursa.

TYPE FEMALE. Body more nearly cylindrical than type male,
9.5 mm. long, 2 mm. in diameter. Tip of proboscis slightly inverted,
size and aramture practically identical with that described for male.
Body filled with developing embryos surrounding and covering
almost all of internal organs. Hard shelled embryos within type
female rather variable in size, 40 to 60% long by 12 to 20» in
diameter.

Type host, the flicker, Colaptes auratus, in intestine. The four
specimens upon which the species is founded were collected at Bowie,
Maryland, October 9, 1906, by Dr. B. H. Ransom.

MORPHOLOGY. The males of this species display a phenomenal
transparency of body structures so that in stained whole mounts
minute internal structures are observable with the greatest ease.
The proboscis in the preserved specimens is so inserted that it
points ventrally at an angle of approximately 60 degrees from the
chief axis of the body. At the base of the proboscis the body proper
is slightly expanded to form a thickened rim. The proboscis recep-
tacle is a cylindrical sac composed of two muscular layers. The
retinacula arise in the brain as two fairly small fibres. Upon pene-
tration of the wall of the receptacle their size increases appreciably.
This increased size is maintained through the remainder of their
course to their insertion upon the body wall. The means of inser-
tion upon the wall of the body is shown in Fig. 4. The lemnisci
are slightly longer than the proboscis receptacle and of smaller
diameter throughout.

26 H. J. VAN CLEAVE

The two testes of the male are roughly oval, lying in the anterior
region of the body. Ltthe (1911:26) described six closely com-
pacted cement glands as characteristic of the genus Plagiorhynchus.
P. formosus appears to have six of these glands (Fig. 4), but their
shape differs radically from the shape of the cement glands of P.
lanceolatus.

The hooks upon the proboscis present a perfect pattern in their
exact arrangement in alternating rows. Roots of the hooks are not
distinct. The base of each hook for a length of about 0.021 mm.
lies embedded in the wall of the proboscis perpendicular to its sur-
face. The free portion of each hook is directed backward at an
angle of about 110 degrees with the basal portion. The foregoing
description of hooks applies to all hooks upon the proboscis except
one or two hooks at the base of each row, which are simple, thorn-
like and one or two of those at the tip of the proboscis which differ
from the remaining hooks in shape and general proportions (see
Fig. 5). In all cases measurement of hooks here as in other papers
by the writer the length of a hook is considered as the straight line
connecting the tip of the hook with its extreme basal part.

IV. THe Genus PotyMorPuHus Lithe, 1911

Acanthocephala parasitic as adults in the intestine of birds.
Posterior tip of the body rather broadly truncated. Anterior end
of body swollen and separated from more attenuated posterior
region by a constriction. Anterior region of the body spined. Pro-
boscis usually cylindrical, frequently smaller at base.

Polymorphus obtusus nov. spec.
Plate II, Figs. 8, 9, 10, 11, and 12

Body with a slightly enlarged anterior region. This enlarge-
ment is set off by a constriction back of which the body again
assumes a diameter equal to that of the anterior enlargement, then
gradually decreases in size to the posterior tip which is more or
less flexed ventrally. Posterior tip terminates bluntly (Fig. 11).
Females 7.7 mm. long, males 4 to 5.5 mm. long. Proboscis in all
specimens examined partially inverted, armed with about sixteen
longitudinal rows of hooks. Anterior body region armed with small

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 27

cuticular spines about 24p long (Fig. 9). Embryos within body of
female 60 to 80u long and 20 to 24» wide with one conspicuous
outpocketing of the middle membrane at each pole (Fig. 10).

Type host Anhinga anhinga (water-turkey), in intestine. Type
locality, Florida. Described from three specimens, one female and
two males. Cotypes in U. S. National Museum, catalog number
2967.

Polymorphus sp?
Plate II, Fig. 13
In addition to Polymorphus obtusus another representative of
this genus has been studied by the writer, but the single specimen,
a male, is insufficient to permit of a description of the species.
Record is given here with a drawing of the proboscis in order to
facilitate comparison in case other specimens are found from the
same host later. The host of this undescribed species in the hooded
merganser, Lophodytes cuculatus. Specimen in the U. S. Bureau
of Animal Industry Collection, catalog number 2808.

V. THe GENUS CENTRORHYNCHUS Lie, 1911
Centrorhynchus spinosus Van Cleave, 1916

Plate III, Figs. 14 and 15
A single species of this genus has been described from North
America. The writer (1916b) described C. spinosus from the intes-
tine of the egret, Herodias egretta. For description see paper cited
above.

VI. THe Genus MepiorHyNCHUS Van Cleave, 1916
Mediorhynchus papillosus Van Cleave

Plate III, Figs. 16, 17, 18 and 19
Mediorhynchus robustus Van Cleave, 1916

Plate IV, Figs. 20 and 21
This genus, which to the present time has been recognized
only from North America, was created by the writer (Van Cleave

1916b) for three new species of Acanthocephala belonging evidently
near to the genus Centrorhynchus, but having characteristics which

28 H. J. VAN CLEAVE

prevented including them in that genus. At the time the description
of this genus was published the writer failed to designate which of
the species should be considered as type. M. papillosus is hereby
designated as type of the genus Mediorhynchus.

Mediorhynchus papillosus has been recorded from Myiochanes
virens (the wood pewee) and Porzana carolina (the sora). Medi-
orhynchus robustus has been found in Icteria virens (the yellow-
breasted chat). |

Mediorhynchus grandis was originally included within this same
genus but a more thorough study of Kostylew’s articles dealing with
the genus Heteroplus has convinced the writer that the species
grandis is, in reality, a member of the genus Heteroplus. Kosty-
lew (1913: 532) considered Heteroplus as near to the genus Gigan-
torhynchus from which he claimed to have separated it. On the
other hand de Marvel (1905: 217) considered Echinorhynchus otidis,
one of the species upon which Kostylew founded his genus Hetero-
plus, as a synonym for E. aluconis. E. aluconis, through the care-
ful work of Lithe (1911), became type of the genus Centrorhynchus.
At the same time Gigantorhynchus mirabilis de Marvel, which Kosty-
lew claimed also belonged to his genus Heteroplus, is clearly, on
the basis of de Marvel’s descriptions and figures, one of the Cen-
trorhynchide. De Marvel’s figures show clearly the constriction
of the proboscis at the line of insertion of the receptacle with the
coincident differentiation of hooks on the anterior and posterior
regions of the proboscis. Kostylew (1913), in describing the pro-
boscis receptacle of Heteroplus, referred to the anterior half as
being twice the diameter of the posterior half. This condition is
typical of the genus Mediorhynchus and serves as an additional
argument for considering the genus Heteroplus as a member of the
family Centrorhynchide and not of the Gigantorhynchide.

VII. THe Genus HeEtTeropius Kostylew, 1914

With the characteristics of the family Centrorhynchide. An-
terior and posterior regions of the proboscis bearing widely differ-
ent numbers of longitudinal rows of hooks.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 29

Heteroplus grandis (Van Cleave, 1916)
Plate V, Figs. 27, 28 and 29
Synonym, Mediorhynchus grandis Van Cleave, 1916

This species, the description of which is given in Van Cleave
1916 b: 226, was described from Quiscalus quiscula (the purple
grackle) and Sturnella magna (the meadow lark). More recently
the writer has examined a specimen from Corvus brachyrhynchus
(the crow) taken in Maryland which proves to belong to this same
species.

Kostylew’s contention that the genus Heteroplus belongs to the
Gigantorhynchide caused the writer at the time of the description
of this species to believe that its superficial and general resemblance
to Heteroplus was not of great significance.

VIII. Tue Genus Firicotiis Lihe, 1911
Filicollis botulus Van Cleave, 1916
Plate V, Figs. 30, 31, 32, 33 and 34

But a single species of this genus, F. botulus, has been recorded
from North America. This species differs widely from F. anatis
of Europe, especially in the shape of the proboscis of the female.
Specific characteristics upon which this species was founded are
given in an earlier paper (Van Cleave 1916). Since the species was
described the writer has discovered younger males than he had seen
at the time of the original description. Figure 30 shows the arrange-
ment of the internal organs of one of these young males. The eider
ducks, Somateria dresseri and S. mollissima, are the known hosts
of the species.

IX. THe Genus ARHYTHMORHYNCHUS Liihe, 1911
Arhythmorhynchus uncinatus (Kaiser, 1893)
Arhythmorhynchus trichocephalus (R. Leuckart, Kaiser, 1893)
Arhythmorhynchus brevis Van Cleave, 1916
Plate IV, Figs. 22, 23 and 24
Arhythmorhynchus pumilirostris Van Cleave, 1916
Plate IV, Figs. 25 and 26

Kaiser in 1893 published a description of Echinorhynchus un-
cimatus Kaiser and E. trichocephalus R. Leuckart. The writer

30 H. J. VAN CLEAVE

(1916a: 172) showed that these two species belong in reality to the
genus Arhythmorhynchus and in the same paper described two addi-
tional species for this genus, A. pumilirostris and A. brevis, both
from the intestine of the American bittern, Botaurus lentigenosus.

X. Species INQUIRENDZ AND SPECIES OF DouBTED DETERMINATION
Echinorhynchus pici collaris Leidy, 1850

Leidy described and recorded the occurrence of four Acantho-
cephalans from North American birds, but no one of these is the
present writer able to locate with any degree of certainty within
the modern system of classification of the group. The descrip-
tion of Echinorhynchus pici collaris from “Picus colaris” is lacking
in details sufficient to permit of speculation as to even the genus
to which it belongs. Consequently E. pici collaris must be regarded
as a species inquirenda. The other three species, given the names
of known European species, probably belong to the respective genera
now recognized as including the European forms.

(?) Echinorhynchus caudatus Zeder of Leidy 1887

This species, which Leidy recorded from Elanoides forficatus
and from Scotiaptex nebulosa, has been considered by the present
writer (1916b:222) as belonging to the family Centrorhynchide,
though the original description is insufficient to permit of a closer
determination. It is extremely improbable that Leidy’s determina-
tion of this species is correct.

(?) Echinorhynchus striatus Goeze of Leidy 1856

From the intestine of Mycteria americana (“Tantalus locula-
tor’), the wood ibis, Leidy recorded the occurrence of Echinorhyn-
chus striatus Goeze. Lthe (1911) was unable to place Goeze’s
E. striatus in his revised system of classification. It seems improb-
able that Leidy was dealing with the species which Goeze described
from central Europe. Many points of detail have caused the pres-
ent writer to consider the form which Leidy recorded as probably
one of the species of Arhythmorhynchus. Its relations to other
species of this genus cannot be determined.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 31

(?) Echinorhynchus hystrix Bremser of Leidy 1887
Leidy’s report of Echinorhynchus hystrix from Anhinga an-
hinga (the water turkey) gives some ground for attributing the
genus Corynosoma as parasitic upon that species of bird, though
here again the specific identity with the European form is to be
seriously doubted.

XI. DistRIBUTION OF THE ACANTHOCEPHALA OF BIRDS

It seems worth while to call attention to the fact that in the
collections from birds examined by the writer the occurrence of
two different species of Acanthocephala within the same host indi-
vidual has never been observed. Furthermore there is no positive
case on record wherein two different genera of Acanthocephala
have been found in the same species of North American bird. The
case of Anhinga anhinga seems to be an exception to this last state-
ment. From this host the writer has described a species of Poly-
morphus while Leidy has described what seems to be a species of
Corynosoma. However in general body form these two genera
resemble one another closely enough to confuse the casual observer.
On this basis the apparent exception to the general condition may
not be a real one.

De Marval, in his significant monographic contribution to the
study of Avian Acanthocephala, has unfortunately failed to furnish
us with any considerable body of data upon the geographical dis-
tribution of the species with which he worked. Evidently many
of his records are compilations from earlier writers. In his treat-
ment of each species of Acanthocephala he has given a host list,
but offers no information as to the locality from which the hosts
were taken. A study of his data shows that he has recorded the
occurrence of Acanthocephala in more than forty families of birds
representing eleven of the seventeen orders of birds recognized
in North America and in addition some families not represented in
the North American fauna.

In the collections available to the writer these parasites have
been found in but seven orders, and a total of but ten families of
birds. Beyond this Leidy’s reports indicate the presence of Acan-
thocephala in at least three additional families. It is a noteworthy

32 H. J. VAN CLEAVE

fact that the geographical distribution of the species known to North
America is restricted almost exclusively to the eastern part of the
continent. In the opinion of the writer this apparent localization
of infestation is probably due to the fact that records from the
West are wanting rather than that the actual distribution is so nar-
rowly limited. Much of the materials studied by the writer has
been the result of the careful work of Mr. Albert Hassall.

XII. DistTRIBUTION OF GENERA OF ACANTHOCEPHALA IN FAMILIES
OF EUROPEAN AND NorRTH AMERICAN BIRDS:

GENERA OF ACANTHOCEPHALA REPORTED FROM

FAMILY OF BIRDS
ACTING AS HOST

CENTRAL EUROPE
(Lithe 1911)

NORTH AMERICA

Colymbide Filicollis
Laridz Filicollis Plagiorhynchus
Anhingide Polymorphus
?Corynosoma
Anatide Polymorphus Polymorphus
Filicollis Filicollis
Corynosoma Corynosoma
?Centrorhynchus?
Ciconiide ?E. striatus?
Ardeide Filicollis Centrorhynchus
?Arhythmorhynchus? Arhythmorhynchus
Rallide Polymorphus Mediorhynchus
Filicollis
Scolopacide Plagiorhynchus
Arhythmorhynchus
Filicollis
Charadriide Plagiorhynchus
Polymorphus
Buteonide ?E. caudatus?
Falconide Centrorhynchus
Strigide ?E. caudatus?
Picide Plagiorhynchus
?E. pici collaris?
Tyrannide Mediorhynchus
Corvide Plagiorhynchus
Heteroplus
Icteride Heteroplus
Mniotiltide Mediorhynchus

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 33

In the above table genera and species marked with a question
mark (?) are of questioned original determination or are species
inquirende. It is quite a striking fact that in only the family An-
atide and probably the Ardeide are any of the same genera of
Acanthocephala found in both North America and Europe.

Lithe (1911), in his register of the Acanthocephala and para-
sitic flatworms from central European hosts recorded the occurrence
of Acanthocephala from hosts representing but eight families of
birds. The accompanying table based upon Luhe’s data and records
of the writer shows in striking manner the differences between the
types of hosts characteristic of the various genera in Europe and
North America. To the writer this table furnishes still farther
evidence of the independence of the acanthocephalan fauna of the
two continents. In the majority of cases even the genera of these
parasites have found hosts in entirely different Families and even
Orders of birds.

XIII. GENERA OF ACANTHOCEPHALA WITH THE ORDERS OF BIRDS
From WHIcH TAKEN

GENUS OF ORDER OF BIRDS SERVING AS HOST
ACANTHOCEPH ALA CENTRAL EUROPE NORTH AMERICA
(Lithe)
Arhythmorhynchus Limicole Herodiones
Centrorhynchus Raptores Herodiones
Anseres
Corynosoma Anseres Anseres
?Steganapodes
Filicollis Anseres Anseres
Pygopodes
Longipennes
Herodiones
Paludicolz
Limicole
Heteroplus Passeres
Mediorhynchus Passeres
Paludicole
Plagiorhynchus Limicolze Pici
Longipennes
Passeres
Polymorphus Limicolz Steganapodes
Anseres Anseres

Paludicolz

34 H. J. VAN CLEAVE

A comparison of infestation within the Orders of birds fur-
nishes a contrast between the two continents even more striking
than that found in the comparison of the Families of birds. The
accompanying table shows among other things, that Corynosoma,
Filicollis and Polymorphus are the only genera having hosts within
the same orders of birds on the two continents.

XIV. Key To THE GENERA AND SPECIES OF ACANTHOCEPHALA OF
; NortH AMERICAN Brirps
(12) Acanthocephala with body proper devoid of spines.............. 2

—

2 (5) Proboscis receptacle inserted at base of proboscis................
bc Pea basse olee gH ee RL easy DD cate SAG batty Genus PLAGIORHYNCHUS.. 3

3). 4): Twenty-four longitudinal rows of hooks: j.\....0....). i245 «00cm
apres ch 2 4 rats. ER Vn ee Plagiorhynchus rectus. Linton

4. (3))-Sixteen) longitudinal sows ‘of ‘hooks. 55... <- 4s. +,«0 ose sree ene meee
ath aie ete fora a poe ere RT Tesret sare cokes Plagiorhynchus formosus nov. spec.

5 (2) Proboscis receptacle inserted at or near middle of proboscis. Pro-
boscis hooks anterior to and posterior to insertion conspicuously
Ney sia od ss ie opts « 3/5) fe ve Glas ol PaaS ie n/a 6.4 sl 6 se 6

6 (7) Invertors of proboscis extending through posterior tip of proboscis
receptacle and continuing as retractors for receptacle...........
BARNS Co AS iS se aie arate avs Rata escete aasle usraan ereneeC ears Genus CENTRORHYNCHUS

Centrorhynchus spinosus Van C., only known species in North America.

7 (6) Invertors of proboscis passing through wall of proboscis receptacle
near its middle or at least considerable distance anterior to the

POSEECION ETI) ooo Peds se ie olam clelerece eieloile wielalni aun he tele nena eee 8
8 (9) Anterior and posterior regions of proboscis bearing different num-
bers of longitudinal rows of hooks............ Genus HETEROPLUS

Twelve longitudinal rows of hooks on the interior region of pro-
boscis, about thirty on posterior. Heteroplus grandis (Van. C.)
only known species in North America.

9 (8) Anterior and posterior regions of proboscis bearing the same num-
ber of longitudinal rows of hooks. Genus MEDIORHYNCHUS. .10

10 (11) Proboscis not covered with conspicuous papillez in which hooks are
embedded. Body usually robust, forms with twenty-four longi-
tudinal rows of hooks on proboscis. Embryos 38u by l6u......

Bh ta iV a a ee Mediorhynchus robustus Van C.

11 (10) Proboscis hooks not conspicuous; embedded in conspicuous papille.

Eighteen longitudinal rows of hooks. Embryos 38 to 47« by
US a tone aoe bo cinerea Mediorhynchus papillosus Van C.

12
13

14
15
16

17

18

19

20
21

22

23

24

25

26

(1)
(14)

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 35

Body proper bearing spines, at least in restricted regions......... 13
Body spines on anterior region of the body and also surrounding
ETAT OPEMMN Orem tis tee lao cera nea eevee Genus CORYNOSOMA, males

But one species, Corynosoma constrictum nov. spec., known to North

(13)
(26)
(23)

(18)

(17)
(20)

(19)
(22)

(21)
(16)

(25)

America.
With no cuticular spines around the genital opening............ 15
Anterior end of the body decidedly larger than posterior end..... 16
Anterior and posterior regions of the body of distinctly different
histological structure............ Genus ARHYTHMORHYNCHUS..17

Hooks on mid-ventral surface of proboscis conspicuously larger.
thanvoneany, Other ParilOL PLrODOSCIS =: /5/4-12 6 aele cision ciaieloieiale ciate
Aleta tetatevelotenersiaeae Arhythmorhynchus trichocephalus (R. Leuckart)
Hooks on ventral surface of proboscis not conspicuously larger
SHAM OM ANY OENEE SUTEACES 8 Le Meals lee cblacaid nia ores eels, eats al glGie is 19
Longest hooks more than: 100m lore! 2465 cise «sale cise cle siv'easeis siete eies
IAI OE 1 AO a pe a Arhythmorhynchus uncinatus (Kaiser)
Longest hooks not more’ than 50 Jong o.oo... iio ee jan a7 simvolescole cals 21
Proboscis with sixteen longitudinal rows of hooks. Embryos 65
to 80u long, 184 wide....Arhythmorhynchus pumilirostris Van C.
Proboscis with eighteen longitudinal rows of hooks. Embryos 76
to 100u long, 24 to 30u wide....Arhythmorhynchus brevis Van C.
Anterior end of body larger than posterior end, but body wall of
same histological structure throughout.............0c.c0e000: 24
Anterior region of body set off from posterior region by conspicu-
ous constriction. Proboscis cylindrical, or frequently tapering
siiehtly toward: bases.2o st ese eee eee! Genus POLYMORPHUS

Polymorphus obtusus nov. spec. only species described from North

(24)

America.

Anterior region of body distinctly swollen, but not separated from
posterior by a constriction. Proboscis spindle shaped..........
MRP Pa Nara ne SCN erayie, eaten a Ne taba re ovat Genus coryNosoMA, females

Corynosoma constrictum nov. spec., only known species from North

America.

(15) Body sac-like or sausage shaped, not conspicuously swollen at ante-

rior end. Proboscis spherical or ovate, followed by a neck fully
as long as the proboscis and sharply set off from the body proper.
BHO Lies CS ches ae eS SRC Re ATE EATEN OLE RY RAP NOV Genus FILICOLLIS

Filicollis botulus Van C. is the only species known to North America.

36 H. J. VAN CLEAVE

XV. SUMMARY

1. This article contains the results of a study of the Acantho-
cephala parasitic in birds from the U. S. Government collections
and the private collections of Professor H. B. Ward and of the
writer.

2. LEchinorhynchus striatus taken by Linton from Ozidemia
americana belongs to the genus Corynosoma and here is described
as Corynosoma constrictum nov. spec.

3. LEchinorhynchus rectus Linton, 1892 is shown to belong to
the genus Plagiorhynchus. P. formosus nov. spec. from Colaptes
auratus is described. These two species constitute the first record
of species of Plagiorhynchus in North America.

4. Polymorphus obtusus nov. spec. is described from Anhinga
anhinga.

5. Polymorphus species? is recorded from Lophodytes cucu-
latus.

6. Mediorhynchus papillosus Van C., 1916 is designated as
type of the genus Mediorhynchus.

7. The genus Heteroplus is one of the Centrorhynchide (not
of the Gigantorhynchide as maintained by Kostylew, its creator).

8. Mediorhynchus grandis VanC., 1916 is shown to belong
to the genus Heteroplus.

9. Corvus brachyrhynchus is cited as a new host for Hetero-
plus grandis (VanC.).

10. Among North American birds the occurrence of two
different species of Acanthocephala within the same host individual
has never been recorded.

11. There is no positive case on record of the occurrence of
two different genera of Acanthocephala within the same species
of North American bird.

12. Tables are given to show the comparison of acanthoce-
phalan infestation in the families and orders of birds of central
Europe and of North America.

13. A key to all described species of Acanthocephala from
North American birds is given.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS af

XVI. LiterRAtTuRE CITED

Kaiser, J. E.
1893. Die Acanthocephalen und ihre Entwicklung. Biblioth. Zool. 7:
1-136.

KostyLew, N.

1913. Ein Beitrag zur Anatomie von Gigantorhynchus otidis Miesch.
Centralbl. Bakt. Abt. 1, 72: 531.

1914. Ueber die Stellung einiger Acanthocephalenarten im System.
Zool. Anz., 44: 186-188.

1915. Contributions a la faune des Acanthocephales de la Russie. Ann.
Mus. Zool. Acad. Imp. Sc., St. Petersburg, 20: 389-394.

Lerpy, J.
1850. Contributions to Helminthology. Proc. A. N. S. Phila., 5: 96-98.

1856. A Synopsis of Entozoa and some of their Ectocongeners ob-
served by the Author. Proc. A. N. S. Phila. 8: 42-58.

1887. Notice of some Parasitic Worms. Proc. A. N. S. Phila., 39: 20-24.

Linton, E.
1892. Notes on Avian Entozoa. Proc. U. S. Nat. Mus., 20: 87-113.

LUue, M.
1904. Geshichte und Ergebnisse der Echinorhynchen-Forschung bis auf
Westrumb (1821). Zool. Annal., 1: 139-250.

1911. Die Siisswasserfauna Deutschlands, Heft 16, Acanthocephalen.
Jena. 116 pp.

DE MarvEL, L.

1905. Monographie des Acanthocephales d’Oiseaux. Rev. Suisse Zool.,
13: 195-386.

Van CLEAVE, H. J.

1914. Studies on Cell Constancy in the Genus Eorhynchus. Journ.
Morph., 25: 253-299,

1916. Filicollis botulus, n. sp., with Notes on the Characteristics of the
Genus. Trans. Amer. Micr. Soc., 35: 131-134.

1916a. A Revision of the Genus Arhythmorhynchus with Descriptions
of Two New Species from North American Birds. Jour.
Parasitol., 2: 167-174.

1916b. Acanthocephala of the Genera Centrorhynchus and Mediorhyn-

chus (new genus) from North American Birds. Trans.
Amer. Micr. Soc., 35: 221-232.

38 H. J. VAN CLEAVE

XVII. ExXPLANATION OF PLATES

All figures excepting Figure 7 are drawn by the aid of a camera lucida
from stained specimens mounted in xylol damar. The magnification is indi-
cated by the projected scale accompanying each drawing.

ABBREVIATIONS USED

b. bursa copulatorix 1. lemniscus
br. brain. p.r. proboscis receptacle
c.g. cement glands r. retinacula
e. egg masses r.p. retractors of proboscis receptacle
ins. insertion of proboscis receptacle t.a. anterior testis
i.p. invertors of proboscis t.p. posterior testis
Prate I.

Figs. 1 to 3, Corynosoma constrictum nov. spec.

Fig. 1. Entire male showing general body shape and arrangement of in-
ternal organs.

Fig. 2. Posterior tip of male showing cuticular spines.
Fig. 3. Embryo from body of mature female.
Figs. 4 to 6, Plagiorhynchus formosus nov. spec.

Fig. 4. Entire male showing general body form and arrangement of inter-
nal organs.

Fig. 5. Profile of the proboscis of same individual as shown in Fig. 4.
Fig. Embryo from body of mature female.
Fig. 7. Plagiorhynchus rectus (Linton). Drawing copied from Linton 1892.

coo)

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS

Imm.

Pate J.

H. J. Van Cleave, del.

H. J. VAN CLEAVE

PuateE II.

Figs. 8 to 12. Polymorphus obtusus nov. spec.

Fig.

Fig.
Fig.
Fig.

Fig.
Fig.

12.
13.

Entire male, poorly preserved specimen, internal organs very in-
distinct.

Cuticular spines in profile, from anterior region of body.

Embryo from body cavity of female.

Posterior extremity of type female showing characteristic blunt-
ness upon basis of which the specific name is given.

Profile of proboscis hooks.

Polymorphus species? from Lophodytes cuculatus.

41

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS

Puiate II.

H. J. Van Cleave, del.

42 H. J. VAN CLEAVE

Prate III.

Figs. 14 and 15. Centrorhynchus spinosus Van Cleave.

Fig. 14. General body form of type female.

Fig. 15. Profile of dorsal surface of proboscis shown in Fig. 14.

Figs. 16 to 19. Mediorhynchus papillosus Van Cleave.

Fig. 16. Entire male showing general body form and arrangement of organs.
Fig. 17. Proboscis and anterior body region of type male.

Fig. 18. Profile dorsal surface of proboscis, same individual as shown in
Fig. 17.

Fig. 19. Embryos from body cavity of fully mature female.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 43

Priate III.

H. J. Van Cleave, del.

44 H. J. VAN CLEAVE

PiatTe IV.

Figs. 20 and 21. Mediorhynchus robustus Van Cleave.

Fig. 20. Male in optical section showing body form and arrangement of
organs.

Fig. 21. Embryos from body cavity of mature female.

Figs. 22 to 24. Arhythmorhynchus brevis Van Cleave.

Fig. 22. Body of entire male.

Fig. 23. Proboscis and anterior region of body of a male.
Fig. 24. Embryos from body cavity of mature female.

Figs. 25 and 26. Arhythmorhynchus pumilirostris Van Cleave.
Fig. 25. Entire male.

Fig. 26. Embryo from body of mature female.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 45

PuateE IV.

H. J. Van Cleave, del.

H. J. VAN CLEAVE

PLATE V.

Figs. 27 to 29. Heteroplus grandis (Van Cleave).
Fig. 27. Proboscis partly invaginated, but showing the hook arrangement

Fig. 28.
Fig. 29.

typical for the genus.
Embryos from body of mature female.
Profile of dorsal surface of proboscis.

Figs. 30 to 34. Filicollis botulus Van Cleave.

Fig. 30.

Fig. 31.
Fig. 32.
Fig. 33.
Fig. 34.

Entire male showing general body form and arrangement of
organs.

Cuticular spines from anterior region of body.
Mature female showing general body form.
Embryo from body cavity of mature female.
Profile, ventral surface, of proboscis of female.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 47

H. J. Van Cleave, del.

Ht Ava ir J
niwoe i ih, hy waht Cay

pl : aN 2 ¢
Hie AU doar 6 aN viel ; fess vie

BRANCHIOBDELLID WORMS (ANNELIDA) FROM
MICHIGAN CRAWFISHES
By Max M. EZttis

Through the courtesy of the Michigan Fish Commission the
writer visited several islands in Potagannissing Bay and Lake Huron
on Patrol Boat No. 4, Captain Robert E. Ellsworth commanding,
during August, 1917. The branchiobdellid worms listed below were
taken from crawfishes collected on this trip and from those of two
additional collections made by Captain Ellsworth. Dr. Walter
Faxon of Harvard University kindly identified the crawfish hosts.

The four species of branchiobdellids represented may be dis-
tinguished by the following key.

a. Anterior nephridia opening to the outside through two separate pores
in segment III; segments VIII and IX each bearing a pair of glandu-
lar disks on the ventral surface; dental formula usually 5-4 or 5-5,

the middle tooth of each jaw being the longest tooth in the jaw.
Xironodrilus formosus Ellis in ed.

aa. Anterior nephridia opening to the outside through one common pore in
the dorsal surface of segment III.
b. Body segments without conspicuously elevated portions; accessary
sperm tube present.

c. Upper and lower lips entire excepting a small, median emargin-
ation; dental formula 5-4, teeth approaching a subequal con-
dition, although the middle tooth of the upper jaw is distinctly
longer than the other four teeth of that jaw.

Cambarincola vitrea Ellis in ed.

cc. Upper lip composed of four, subequal lobes, which can be ex-
tended as digitiform processes; lower lip composed of two,
subequal lobes which can be extended; a small, lateral lobe
on each side at the junction of the upper and lower lips;
dental formula 5-4; middle tooth of upper jaw long and
prominent; lateral teeth small, not more than half the length
of the middle tooth.

Cambarincola philadelphica (Leidy)
bb. Dorsal portions of segments elevated; segments VII and VIII with
funnel-shaped enlargements of the dorsal portions of the seg-
ment, more or less completely encircling the segment; funnel of
segment VIII excavated dorsally so that its dorsal margin bears

two, small “horns”; dental formula 5-4.
Pterodrilus durbini Ellis in ed.

50 MAX M. ELLIS

(1) XzRoNnopRiLus FormMosus Ellis
From Cambarus virilis Hagen
1. James Island, Potagannissing Bay, August 4.
2. Three miles up Potagannissing River, Drummond Island, Pot-
agannissing Bay, August 4.
3. Pilot Harbor, Sitgreaves Bay, north side of Drummond Is-
land, Potagannissing Bay, August 6.
4. Little Cass Island, head of Detour Passage, August 6.
5. Churchville Point, head of Lake George, 46° 31’ N., August 7.
6. Harbor Island, Potagannissing Bay, August 8.
7. Winona Slips, Bay City, Saginaw Bay, September (Capt. Ells-
worth).
(2) CAMBARINCOLA VITREA Ellis
From Cambarus virilis Hagen
1. James Island, Potagannissing Bay, August 4.
2. Three miles up Potagannissing River, Drummond Island, Pot-
agannissing Bay, August 4.
From Cambarus propinquus Girard
1. Sault Sainte Marie, St. Marys River, August 7.
2. Echo Lake, Grand Island, Lake Superior, August 17 (Capt.
Ellsworth).
(3) CAMBARINCOLA PHILADELPHIA (Leidy)

From Cambarus virilis Hagen
1. James Island, Potagannissing Bay, August 4. .
2. Pilot Harbor, Sitgreaves Bay, north side of Drummond Is-
land, Potagannissing Bay, August 6.
3. Little Cass Island, head of Detour Passage, August 6.
4. Harbor Island, Potagannissing Bay, August 8.
(4) PTERODRILUS DURBINI Ellis
From Cambarus virilis Hagen

1. James Island, Potagannissing Bay, August 4.
2. Three miles up Potagannissing River, Drummond Island, Pot-
agannissing Bay, August 4.

3. Pilot Harbor, Sitgreaves Bay, north side of Drummond Is-
land, Potagannissing Bay, August 6.

Little Cass Island, head of Detour Passage, August 6.

Churchville Point, head of Lake George, 46° 31’ N., August 7.

Harbor Island, Potagannissing Bay, August 8.

Winona Slips, Bay City, Saginaw Bay, September (Capt. Ells-
worth).

SOK eu

It may be seen from this list that Xironodrilus formosus Ellis
and Pterodrilus durbini Ellis were found in every collection from

BRANCHIOBDELLID WORMS 51

Cambarus virilis Hagen. In each case Xironodrilus formosus was
the more abundant species, represented by hundreds of individuals.
Comparatively few Pterodrilus durbini were taken. The relative
abundance of these two species may be considered as accurate for
these collections as the living crawfish were dropped into the killing
fluid as soon as caught, and all of the branchiobdellid worms carried
by each crawfish preserved. The two species of Cambarincola, if
found, were represented by a fair number of individuals. Cambarin-
cola vitrea Ellis was the only species taken from Cambarus pro-
pimquus Girard in these collections. Both species of Cambarincola
here represented however have been taken from specimens of Cam-
barus propinquus at Douglas Lake, Michigan.

Umiversity of Colorado.

DEPARTMENT OF NOTES, REVIEWS, ETC.

It is the purpose, in this department, to present from time to time brief original
notes, both of methods of work and of results, by members of the Society. All
members are invited to submit such items. In addition to these there will be given a
few brief abstracts of recent work of more general interest to students and teachers.
There will be no attempt to make these abstracts exhaustive. They will illustrate progress
without attempting to define it, and will thus give to the teacher current illustrations, and
to the isolated student suggestions of suitable fields of investigation.—[Editor.]

A CHART ON GENERAL PLANT HISTOLOGY AND PHYSIOLOGY

The valuable teaching aid afforded by charts and diagrams of
various sorts is well understood by most teachers of biology who
are more or less well acquainted with the numerous current sets of
charts offered to the profession. There are a few teachers who
possess the enviable talent of rapidly constructing excellent black-
board sketches during a given lecture or laboratory period to illus-
trate the particular features or phenomena under study at that
particular time. Some instructors have made use of the more
permanent crayon sketches on sheets of drawing paper hung over
an easel. The uses of the various lantern-slide, opaque, vertical
and micro-projection possibilities are also utilized to a very desir-
able degree under certain circumstances.

The principal pedagogical difficulty which all teachers have prob-
ably experienced in the practical application of these or other useful
adjuncts of the same general kind to teaching is, that at best the
student gets a disjointed presentation of the subject in question for
the reason that the subject matter must be presented more or less
disjointedly and interruptedly because of the many other things
which we compel him to study. The student suffers from this regu-
lar lack of continuity. He loses much because of his failure to see
the real position of a given structure or place of a given activity in
the organism as a whole.

I may illustrate my meaning here by a reference to the common
practice in teaching some of the phases of botany, say phytohistology.
Probably students may be found in every class in plant histology
as that subject is currently taught, who, knowing right well the
detailed characteristics of all of the common tissues of the vege-
table kingdom and how to handle the histological technique involved

54 NOTES, REVIEWS, ETC.

in the preparation of such tissues, may fail utterly to acquire an
adequate knowledge of the place and function of those tissues in
the plant as a whole. In fact such students may not even know
where in the plant to find a given tissue if they are thrown upon
their own resources, resources derived from their histology courses.
Surely it is most difficult for them to really understand the function
of the various tissues of the plant if they do not know the position
of the tissues within the plant.

The same general state of affairs sometimes exists in plant
physiology. The student may understand the various fundamental
processes of the plant very well, but he often fails to read through
the whole series of interrelated activities and to visualize, as it
were, the individual plant as a completely equipped and working
entity.

The most fundamental process in all nature is photosynthesis.
Consequently it is quite desirable that the student of general biology,
of general botany, surely of general physiology and general science
should understand at least the fundamental features and the signi-
ficance of that great phenomenon. He should know photosynthesis
as it occurs in the leaf, of course, but he should also know about
many of the other processes and structures which make the photo-
synthetic manufacture of carbohydrates in the leaf possible. He
must know quite well many of the interrelations of the various activi-
ties of the plant if he wishes to really understand photosynthesis.
He must see the plant as a completely constructed mechanism with
its various parts working together in harmony. It is the business
of the teacher to see to it that he does get this notion definitely out-
lined in his mind.

Now to be sure that our students really get all of this and this
point of view of the plant, requires more than a little thought on
the part of the teacher as to the methods of instruction that are to
be followed in the class-room and laboratory. Sometimes I have
thought that some of the common difficulties in this connection might
be lessened if the class were to take a single common plant, say the
sunflower, castor bean or scarlet runner, and work out its entire
structural and physiological life-history from the embryo in the
seed through the development and maturation of the new individual

AMERICAN MICROSCOPICAL SOCIETY 55

including the new fruit and seed. If the exercises on histology
and physiology were properly correlated at every point possible I
believe that such a method would result in giving to the student an
admirable introduction to many of the fundamentals of general bot-
any. At the conclusion of such a course the student would have
secured an admirable and useful insight into the most of the im-
portant plant phenomena. And I believe that he would actually
retain a definite mental picture of what the plant is as a mechanism
and what it does as a living, working organism. From such a course
he would surely obtain a connected view of these matters and he
would not think of the plant as composed of a group of parenchyma
cells here, a strand of fibers there, an irregular patch of epidermis
somewhere else. He would not stop to wonder how and from
whence the root gets the necessary food for its growth or how it is
possible for growth to begin in a tree in early spring before the
leaves have unfolded their chlorophyll-containing tissues.

Something of the same results may be secured by the skillful
teacher from a carefully planned summary of the whole matter in
which the various interrelations and correlations are plainly worked
out. I have found in my own department that generalized draw-
ings aid greatly in connection with such summaries. An illustration
which brings before the student by means of a single chart or page
many of the essentials has been found most useful by myself and
the other instructors in my department. The late Professor Bessey
was a master in preparing such diagrams. His specialty was, as
all know, the preparation of figures to show lines of descent and
evolution of the groups of the plant world. Whether one could
agree as to the derivations and progress of evolution represented
by his diagrams or not did not detract from the fact that his figures
helped greatly to portray the principles and the theories that he
wished to emphasize. Thousands of his students were enabled to
secure a much better idea as to what evolution really is from such
methods even if they went no further in their study of biology
than the Freshman course.

Figures or diagrams of this sort may be studied indefinitely
and the longer they are examined the more illuminating they become
until the average student is enabled to secure a complete and prop-

THE HISTOLOCY AND PHYSIOLOCY OF THE PLANT

REPRODU a
SS
PRIMARY

SS ‘ik TRANSPIRATION
= ‘ il | i, 20

CUTICLE

eecseyenme

CAMBIUM

ROOT HAIRS
GROWING a

WATER
/ AND SOLUTE
MITROGER
>» FIXATION

K

PLATE VI

AMERICAN MICROSCOPICAL SOCIETY 57

erly balanced conception of the chief features of the subject in
question.

Such figures may be constructed in great detail and with much
technical skill or they may be more or less crude and diagrammatic
and still be of great value to the learner. With the information
gained from careful studies of plant anatomy and plant processes
the student should be able to fill in the missing details of a general-
ized drawing and to interpret properly the diagrammatic features
of the figure. This being possible, a diagram of the kind submitted
herewith should be exceedingly helpful and, indeed, illuminating.
It was solely because of the success of these methods in this depart-
ment that I was led to publish these brief notes and the diagram
in the hope that they might be suggestive in some degree to my
fellow workers in biological fields.

The figure as published here has been revised and redrawn
from a similar figure published in 1914 by the writer in a labora-
tory manual of plant physiology. The older diagram was later
enlarged and worked out in the form of a wall chart. The reader
will understand, of course, as he looks at this diagram that the
relative proportions of the various structures exhibited are not
intended to be represented at all as they actually occur in the living
plant. In fact the proportions are mostly so unnatural as to be
grotesque and even misleading if the student or reader does not
understand how to interpret them in the light of what has been said
in the above paragraphs. He is supposed to have acquired this
understanding in his courses of study dealing with the plant. The
figure merely helps him to see the imterrelations of the facts of his-
tology and physiology graphically summarized in the features of the
chart. The chart represents in a more or less diagrammatic man-
ner an epitome of the great facts of histology and physiology.
Many additional entries might be made upon the chart, but the
danger is in so multiplying details that the real purpose of the
sketch may be obscured behind the maze of unessential details.
That would be a serious blunder in the use of this method of
teaching or learning.

I believe that it is one of the chief duties of a teacher to epi-
tomize very carefully his subject as fully as possible by whatever

58 NOTES, REVIEWS, ETC.

methods he may devise. In so far as he is successful in this prac-
tice so far will the students in his classes carry away with them a
definite and compact and possibly useful body of knowledge upon
the subject taught. Such a chart as this has been found very
useful in such an epitome, and the method in general is successful
in application.

Raymonp J. Poot, Pu. D.
The University of Nebraska.

A METHOD FOR MOUNTING ANATOMICAL PREPARATIONS FOR
; EXHIBITION

Oftentimes students in comparative anatomy make excellent
preparations which are worthy of preservation. But this is not done
as a rule because of the trouble in mounting. A glass strip of just
the right size to fit into the exhibition jar must be found. The
preparation is with difficulty tied by thread to the glass plate. If
one could only “pin” into glass! The following method is sug-
gested as a solution. A mixture of hard paraffin, beeswax and
lampblack is melted up and poured into a paper box cover about
the size needed had a glass plate been used. The mixture in the
cover should be between %4 and % inch deep. It should be allowed
to cool somewhat and then on this bed the preparation should be
placed and pressed down into it somewhat. A few small pins can
easily be made to fasten it securely. When the matrix is cooled
they can be clipped off on the back. Labels can also be easily
attached to parts of the dissection. The entire cover can now be
placed in cold water for a few minutes. When hardened the cover
can be cut away with a knife—the paraffin background cut down
to just the size that will fit into the glass jar—care being taken to
make it fit in snugly. The jar can now be filled with formalin and
the cover fastened on. The black background makes the objects
stand out distinctly and the preparation never becomes loosened
from its wax bed. The whole operation takes but a few minutes
time.

G. Gi Scorn

Department of Biology,

College of the City of New York.

AMERICAN MICROSCOPICAL SOCIETY 59

GREEN LIGHT FOR DEMONSTRATING LIVING CESTODE OVA

By projecting the light from a small arc through two screens,
one of Picric Acid yellow and one of Methyl Blue blue, a green
light was obtained which gave a peculiarly sharp definition to the
details of living Cestode ova. The eggs were studied on a slide
under either low or high power. This light has been used in
connection with the demonstration of the eggs of Dipylidium ca-
ninum, Tenia crassicollis and Hymenolepis nana. The color screens
were prepared by staining lantern slide plates, after dissolving out
the silver with Hypo from the unexposed plate, in either a weak
solution of Picric acid or Methyl Blue. In each case the film
was stained until it showed a distinct color when washed in water.

University of Colorado. M. M. ELtIs.

A NEW METHOD OF STAINING TISSUES CONTAINING NERVES

Captain Sydney M. Cone, M. D. (J. Am. Med. Assn., Jan. 19,
1918) proposes the following method for nerves, which he says is
excellent in bringing out axis cylinder and medullary sheaths, being
approached only by Bielschowsky’s Axis Cylinder stain. The latter
requires days, and does not give contrast staining.

(1) Harden in 4% formaldehyde for 6 days; (2) carry thru
graded alcohols to ether and absolute alcohol; (3) embed in cel-
loidin and cut 10-20 microns, washing in water; (4) stain 15 min-
utes in carbol fuchsin, washing rapidly in water; (5) place in 1%
osmic acid 5 minutes; (6) wash rapidly, and stain for 1 minute in
50% aqueous solution of safranin; (7) place in 1% acid alcohol
1 minute; and in 95% alcohol for 2 minutes; (8) place in absolute
alcohol and clove oil alternately until sections appear deep pink and
translucent; (9) place in xylene 2 minutes, and mount in Canada
balsam.

Paraffin sections sometimes stain fairly well, but it is not safe,
as xylene used to remove the paraffin before staining interferes
with the process. The formaldehyde hardening is essential for the
success of the stain.

[I wonder whether terpineol could not be used here, or berga-
mot, or naphtha, or even toluol. It would be a good thing if labora-

60 NOTES, REVIEWS, ETC.

tory workers would give the newer results for clearing agents that
do not decolorize stains, used after imbedding in (a) celloidin or
parlodion, or (b) paraffin.—V. A. Latuam, Abstractor.]

FONTANA’S SPIROCHETE STAIN

Fontana’s method of staining spirochetes involves the use of
the following preparations:—1l. Fixing fluid: acetic acid, 1 c.c.;
formalin, 20 c.c.; distilled water, 100 cc. 2. Mordant: tannic
acid, 5 gm.; phenol solution (1 per cent), 100c.c. 3. Silver solution:
Prepare a 0.25 per cent solution of nitrate of silver, which may be
done with sufficient accuracy by dissolving a small crystal in half a
test tube of distilled water and adding just enough ammonia solu-
tion to cause a slight permanent turbidity. 4. Distilled water.

3. Process: Prepare the slide to be stained by spreading the
material from the syphilitic lesion very thinly on a clean slide, allow-
ing to dry spontaneously ; fix by pouring on the fixing fluid, pouring
it off after a few seconds. Renew immediately, and perform this
process several times. The total duration of this stage should be
not less than a minute. Wash well in distilled water, flood with the
mordant, apply gentle heat until steam arises, and allow the process
to continue for half a minute; wash thoroughly in distilled water
(15 to 30 seconds), flood with the silver solution, again warm gently
for half a minute, wash, blot and dry. Mount in balsam for per-
manent specimens. The spirochetes are stained jet black, and appear
larger than when stained by ordinary methods. Cedar oil causes
the spirochetes to pale. Vi AL

SIMPLE METHOD OF CLEANING OLD USED SLIDES
Johnson (J. Am. Med. A., Dec. 8, 1917) suggests soaking slides
indefinitely (24 hours at least) in full strength commercial (house-
hold) ammonia, followed by rinsing with water and wiping clean.
Stained smears of all kinds, immersion oil, balsam mounts are
equally well cleaned. The same supply may be used repeatedly if
kept in tightly closed receptacle. V.-AL UE:

MENTHOL FOR NARCOTIZING
Don’t forget that menthol is an effective reagent in narcotizing
or anzsthetizing lower forms of life such as Rotifers, Infusoria, or
even small crustacea. Vo Aris

AMERICAN MICROSCOPICAL SOCIETY 61

FRESH WATER BIOLOGY

Teachers in America have been eagerly awaiting this volume
for several years. The knowledge of the plans of the editors and
publishers, including as they have the collaboration of a large num-
ber of active biologists, has inevitably become wide spread. The
need of such a work, planned on a scale that would be at once
liberal and feasible, has been so genuine that it was assured in
advance of a very wide use among teachers and working biologists.

The unanimous verdict will be that every reasonable expecta-
tion has been met by Professors Ward and Whipple and their
helpers, in spite of the fact that the delays inevitable in getting
such a synthetic task before the public will cause many of the
contributors themselves to feel the need of revising their work by
the time of its first appearance. All teachers of Biology, all
advanced students of any group, all amateurs who use the micro-
scope on living things, will find “Fresh Water Biology” a necessary
part of their equipment.

The volume is, in many ways, very close to the kind of work
which has long been fostered and advanced by the American Micro-
scopical Society and its Transactions. An organized interest in
limnological work was manifest as early as 1899, at which meeting
a Limnological Commission, consisting of Professors Birge, Eigen-
mann, Kofoid, Ward and Whipple, was appointed to “unify, extend,
and stimulate limnological work in this country.” The following
year this Commission made a report which anticipated much of
the ecological work done since with the fresh-water forms of this
country, and unquestionably gave inspiration and impetus to the
studies on which this book is based. While the volume cannot be
listed among the annual “Transactions” of the Society, certain it
is that much of the contributory work leading to this fine showing
in American fresh-water Biology has been done by members of
this Society and published in one form or another in its Transac-
tions.

So close is this enterprize to what this Society has been encour-
aging in every possible way for many years, that the pages of the
Transactions are now freely offered the editor and collaborators,

62 NOTES, REVIEWS, ETC.

pending new editions of the book, for such supplementary and
revisional statements as may be necessary from time to time to
keep the accounts and keys of the various groups up to date. Such
a cooperative arrangement would contribute greatly both to the
convenience of our membership and to the most effective use of
this manual.

The work is much too compendious and condensed to allow an
adequate statement even of its scope, much less to bring to our
readers any of its specific contents. In general the material pre-
sented is to be classified under three headings: (1) General dis-
cussion of the conditions of life and of the effective study of organ-
isms; (2) the biological conditions, method of collection, culture
and preservation of the special groups; and (3) systematic keys,
with descriptions and illustrations of the classes, orders, families,
genera, and representative American species of the groups treated.

Under the first head may be included the introductory chapter
by Professor Ward, the chapter on “Conditions of Existence” by
Professor Shelford, on “Methods of Collecting and Photograph-
ing” by Professor Rieghard, and the concluding chapter by Pro-
fessor Whipple on ‘“‘Technical and Sanitary Problems,” as related
to fresh waters. The biological features of the special groups are
treated at the beginning of the appropriate chapters. The chap-
ters on Bacteria, Larger Aquatic Vegetation, and Aquatic Verte-
brates are confined to this aspect, making no effort at systematic
display.

The following experts furnish the systematic chapters: Ed-
gar W. Olive, Blue-green Alge; Julia W. Snow, Other Fresh
Water Alge; C. H. Edmondson, Amceboid Protozoa; H. W. Conn
and C. H. Edmondson, Flagellate and Ciliate Protozoa; Edward
Potts, The Sponges; Frank Smith, Hydra and Other Fresh Water
Hydrozoa; Caroline E. Stringer, The Free-living Flatworms ; Henry
B. Ward, Parasitic Flatworms; Wesley R. Coe, The Nemerteans;
N. A. Cobb, Free-living Nematodes; H. B. Ward, Parasitic Round-
worms; H. S. Jennings, The Wheel Animalcules; H. B. Ward,
Gastrotricha; Frank Smith, Aquatic Chetopods; J. Percy Moore,
The Leeches; A. S. Pearse, The Fairy Shrimps; E. A. Birge, The

AMERICAN MICROSCOPICAL SOCIETY 63

Water Fleas; C. Dwight Marsh, Copepoda; R. W. Sharpe, Ostra-
coda; A. E. Ortman, Higher Crustaceans; R. H. Wolcott, The
Water Mites; James G. Needham, Aquatic Insects; Charles B.
Davenport, Moss Animalcules; Bryant Walker, The Mollusca.

Two devices in the arrangement of the systematic matter call
for comment. The guide numbers in the artificial keys are arranged
in accordance with a plan developed by Professors Forbes and
Smith at the University of Illinois. Each guide line begins with
a number. In addition to its own appropriate number which leads,
there follows in parentheses the alternative number (or numbers)
which indicates the contrasted line to which the seeker must go if
that particular legend is not diagnostic. This is true both of the
earlier and the later guide lines in a given series. If a given key
line is acceptable the further guiding number is at the close of the
line. The device thus gives a perfect system of cross references
both forward and backward between categories of a given grade.
This is unnecessary in brief keys; but where there are scores of
intervening subordinate categories it is a great convenience. The
name and description of a species, all the supplementary biological
facts concerning it and the illustration are included in a solid panel
between its own key line and the next. This gives a convenient
compactness which is very satisfying.

The general impression which follows examination of the book
is the perfectly enormous amount of material condensed into its
somewhat more than 1000 pages. This means, of course, great
brevity, and yet no one interested in these groups can feel that the
interesting and important matter has been left out. To one whose
studies are confined largely to a single group there must come
a renewed and enlarged sense of the representative character of
the fresh-water organisms. One has brought home to him also
the vast incompleteness of our records of the American distribution
of even the better known fresh-water species. It ought to be pos-
sible in connection with the extended use and further revision of
such a work as this to get a better account of specific range in this
country.

It seems ungenerous to mention slight imperfections where
so much has been brought to our aid. However, the appearance

64 NOTES, REVIEWS, ETC.

of the chapters on Protozoa and Oligochztes is marred by the use
of occasional cuts too heavy and opaque to give any true idea of
the delicacy of the organisms. Figure 982 of Chetogaster is an
example of this.

A list of important references, in no case purporting to be a
complete bibliography, concludes each chapter. An adequate index,
including important descriptive terms and all of the scientific names
used in the keys, concludes the book.

Fresh Water Biology, by Henry B. Ward and G. C. Whipple, with a staff of
Specialists collaborating. Pages ix and 1111, with 1547 text figures. John Wiley and
Sons, New York and London, 1918. Price, $6.00.

AN INTRODUCTION TO THE HISTORY OF SCIENCE

Nothing which has come to the attention of the reviewer puts
more convincingly the meaning of the history of science than the
preface of this little book by Professor Libby. “The history of
science has something to offer to the humblest intelligence. It is a
means of imparting a knowledge of scientific facts and principles
to unschooled minds.

“The history of science is an aid in scientific research. It
places the student in the current of scientific thought, and gives
him a clue to the purpose and necessity of the theories he is required
to master. It presents science as the constant pursuit of truth
rather than the formulation of truth long since revealed; it shows
science as progressive rather than fixed, dynamic rather than static,
a growth to which each may contribute.

“It is only by teaching the sciences in their historical develop-
ment that the schools can be true to the two principles of modern
education, that the sciences should occupy the foremost place in
the curriculum and that the individual mind in its evolution should
rehearse the history of civilization.

“The history of science should be given larger place than at
present in general history. History of science studies the past
for the sake of the future. It is a story of continuous progress.
It is rich in biographical material. It shows the sciences in their
interrelations, and saves the student from narrowness and pre-
mature specialization. It affords a unique approach to the study

AMERICAN MICROSCOPICAL SOCIETY 65

of philosophy. It gives an interest in the applications of knowledge,
offers a clue to the complex civilization of the present, and renders
the mind hospitable to new discoveries and inventions.

“The history of science is hostile to the spirit of caste. It
reveals men of all grades of intelligence and of all social ranks
co-operating in the cause of human progress. It is a basis of intel-
lectual and social homogeneity.

“Science is international,—English, Germans, French, Italians,
Russians—all nations—contributing to advance the general interests.
[The teaching of it] cannot fail to enhance in the breast of every
young man or woman faith in human progress and good will to all
mankind.”

In method, this introduction takes up certain great scientific
relations and applications, and treats these largely in connection
with the personality of the men who have contributed their solu-
tions. Some of the graphic chapter headings will carry the sug-
gestion of method and of content:—1. Science and Practical Needs
—Egypt and Babylonia; 2. Influence of Abstract Thought—Greece:
Aristotle ; 3. Scientific Theory Subordinated to Application—Rome:
Vitruvius; 4. The Continunity of Science—the Medieval Church
and the Arabs; 6. Scientific Method; 7. Science as measurement;
8. Cooperation in Science; 9. Science and the Struggle for Lib-
erty; 10. Interaction of all the Sciences; 11. Science and Religion;
12. Reign of Law; 14. Scientific Prediction; 16. Science and War;
17. Science and Invention; 19. The Scientific Imagination; 20.
Science and Democratic Culture.

The presentation is simple, direct, vivid, untechnical, and well
suited to the intelligent reader with general interests.

An Introduction to the History of Science, by Walter Libby. [Illustrated; 288
pages. Houghton, Mifflin Company, Boston, 1917. Price, $1.50, postpaid.

A SHORT HISTORY OF SCIENCE
Evidently the stay and the work of M. Sarton in this country
is helping create an atmosphere in which we may prophesy an exten-
sion of interest in the history of science. In this atmosphere our
own American teachers, who have been doing something in this field
for their students, are being encouraged to bring their work to the

66 NOTES, REVIEWS, ETC.

more general audience. All this is very much worth while and will
stimulate the giving of similar courses in many of our schools and
colleges, both to culture the general student and to unify the scien-
tific consciousness of the student of science.

The book under review is by Professors Sedgwick and Tyler
and embodies very largely the well known course of lectures on
the subject begun by the senior author in Massachusetts Institute
of Technology more than twenty-five years ago. The purpose is
expressed by the authors thus,—“To furnish a broad general per-
spective of the evolution of science, to broaden and deepen the
range of the students’ interests, and to encourage the practise of
discriminating scientific reading, . . . by furnishing the student
and the general reader with a concise account of the origin of that
scientific knowledge and that scientific method which, especially
within the last century, have come to have so important a share in
shaping the conditions and directing the activities of human life.”

The general treatment is broadly chronological and geographic,
—following the origin and rise of the wonderfully varied civiliza-
tions of the near-Mediterranean peoples and their distinctive marks
upon the progress of knowledge, of its applications, and of the
method and spirit which its right pursuit demands of its followers.
The chapter headings indicate this phase of the treatment: Early
Civilizations; Early Mathematical Science in Babylonia and Egypt;
Beginnings of Science; Science in the Golden Age of Greece; Greek
Science in Alexandria ; Decline of Alexandrian Science; The Roman
World,—The Dark Ages; Hindu and Arabian Science; Progress
to 1450 A. D.; A New Astronomy and the Beginnings of Modern
Natural Science ; Mathematics and Mechanics in the Sixteenth Cen-
tury; Natural and Physical Science in the Seventeenth Century ;
Beginnings of Modern Mathematical Science; Science in the Eigh-
teenth Century; Modern Tendencies in Mathematical Science; Ad-
vances in Science in the Nineteenth Century.

Within these general headings, further analysis and presenta-
tion are based upon a combination of biography, the rise and solu-
tion of problems, and the discovery of the principles which have
proved significant and fruitful. Topics like the following raise
the expectations of the reader and indicate the emphasis: Primitive

AMERICAN MICROSCOPICAL SOCIETY 67

Interpretations of Nature; Astrology; Primitive Counting and
Geometry ; Mathematics in Egypt; the Calendar and Measurements
of Time; Greek Mathematics; Pythagoras; Beginnings of Rational
Medicine; the Hippocrates; the Sophists; Circle Measurements ;
Aristotle; Euclid; Archimedes; Earth Measurements; Beginnings
of Human Anatomy; Mathematics and Astronomy at Alexandria;
Ptolemy ; Hindu Astronomy; Arabian Contributions to Mathematics
and Astronomy; Renaissance and Sciences ; Alchemy ; the Compass ;
Clocks; Textiles; Printing; the New Astronomy,—Copernicus,
Tycho Brahe, Kepler, Galileo; Medicine and Chemistry, Anatomy ;
Vesalius ; Higher Algebraic Equations and Symbolic Algebra; Gre-
gorian Calendar; Harvey and Blood Circulation; Studies of the
Atmosphere, Barometer, gases; Phlogiston; Beginnings of Chem-
istry ; Bacon and Descartes; and thus on to the great wave of mathe-
matical and natural science discoveries of the eighteenth and nine-
teenth centuries which cannot even be enumerated here.

The book is enlivened thruout by appropriate quotations from
the men who did the work and from appreciative commentators on
that work. In a series of appendices are more lengthy documents,
—as, the oath of Hippocrates, Dedications by Copernicus and Har-
vey, Gallileo before the Inquisition, and the like. Appendix I
enumerates and discusses briefly some leading inventions of the
last two centuries.

The volume closes with a table of the important dates in the
history of science and of civilization, a brief list of reference books,
and an index. Each chapter closes with a list of references.

The book is attractively made up and printed.

A Short History of Science, by Sedgwick and Tyler. Illustrated, 474 pages.
The Macmillan Co., New York, 1917. Price, $3.50.

BIOCHEMICAL CATALYSTS IN LIFE AND INDUSTRY
This volume discusses only the proteolytic enzymes, being the
second volume by the author on enzymes and their uses. A pre-
liminary chapter discusses the nature of the transformations that
take place in the living cell, the inorganic catalysts, the biochemical
catalysts, the theories as to their mode of operation, and a classifi-
cation of proteolytic enzymes based on the number of molecules of

68 NOTES, REVIEWS, ETC.

water they are capable of fixing in a molecule of albumin. Follow-
ing Schutzenberger’s conception of the structure of the polypeptide
molecule, the author presents a very attractive and cogent statement
of the mechanism of progressive hydrolysis of these molecules under
ferment action.

The general discussion proceeds under these heads:—The
Coagulating Catalysts,—thrombin, myosinase, and rennet; Pepsin;
Trypsin, both pancreatic and from various animal and vegetable
sources; Erepsins, including those secreted in the intestines, the
poorly defined peptolytic enzymes which act on so-called peptones,
nucleases which transform the phosphoric nucleo-proteins, argin-
ase, and a small group of creatin-destroying catalysts; and the Amz-
dases, the group of enzymes which aid in the final decomposition
of the amino-acids,—the last stages of the reduction of the protein
molecule before assimilation or excretion.

The statement of the nature, origin, mode of isolation, prop-
erties, and physiological role of these vital substances is extremely
lucid, and meets the need of the general biologist who has not
the opportunity to keep abreast with the more technical aspects of
this department of biochemistry.

Most general readers will be especially attracted to Part VI,
which deals with the applications of these organized catalysts to
medicine and industry, together with the grounds upon which such
applications are possible. The author traces the use and abuses of
pepsin in therapeutics, and progress made in standardizing tests of
its efficiency both as to dissolving and in actual peptonizing power.
Reference is made to peptones, both peptic and pancreatic, offered
as an easily assimilable diet for greatly debilitated patients. Sim-
ilar preparations are used in making culture broths in bacteriolog-
ical laboratories.

In a similar way diagnosis of stomach states is made by analysis
of the gastric contents at different stages of test meals, with a view
to obtaining the amount of chemical change, the acidity, and the
enzymic contents. The author holds that the disrepute into which
this determination has fallen is due to poor methods of application
rather than to any fault of the principle itself.

In preservation and use of grains and flours native proteolytic

AMERICAN MICROSCOPICAL SOCIETY 69

enzymes, and those produced by micro-organisms on the surface
of the grains or placed in the flour purposely, bring changes that
must be considered. So in brewing and in grain distillation, these
biological catalysts play an essential role. The same processes are
seen in the milk ferments and in the ripening of cheeses. In the
latter some of the enzymes are native to the milk, some are pro-
duced by micro-organisms, and rennet is added artificially.

There is an interesting discussion of the relation of the pro-
teolytic milk ferments to intestinal putrefaction. The writer him-
self has done work with the Bulgarian ferment, and his views
as to the cause of the benevolent intestinal action of the various
clotted milks are contrasted with those of Metchnikoff and others.

Other topics discussed are :—putrefaction, enzymes operative in
tanning, biocatalysts of the soil, assimilation of atmospheric nitro-
gen, fertilizers, recovery of nitrogenous wastes, and artificial nitro-
genous foods.

As the outcome, largely, of his own experiments the author
sums up his conclusions in respect to the last item thus:—“It ap-
pears that there is ample proof that the organism draws all its
nitrogenous constituents from the hydrolysis of proteins. These
may result either from the actual process of digestion, or from
artificial means, like the action in vitro of proteolytic enzymes or
the action of concentrated acids. In all events, these [artificially
reduced nitrogen molecules] are directly assimilable substances and
should be considered as food materials of great nutritive value.

In fact, it has been established that a mixture of amino-acids,
containing qualitatively and quantitatively all the principal products
of the complete hydrolysis of proteins, can replace the albuminoid
foods, and as such maintain the animal organism in nitrogenous
equilibrium.” The writer is convinced that nutrition can ultimately
be effected more economically and rationally by the substitution of
some of these artificially produced nitrogenous foods for the com-
plex natural ones, such as meat.

Each chapter is followed by a bibliography ; and an index closes
the book. The mechanical part is well done.

Biochemical Catalysts in Life and Industry, by Jean Effront. Translated by

Samuel C. Prescott, 752 pages. John Wiley and Sons, New York, 1918. Price, $5.00,
postpaid.

PROCEEDINGS

of the American Microscopical Society
MINUTES OF THE PITTSBURG MEETING

The thirty-sixth annual meeting of the American Microscopical Society
was held in affiliation with the A. A. A. S. at Pittsburg, Pa., Dec. 29, 1917.

In the absence of President Guyer, Vice-President Griffin acted as
chairman.

The report of the Custodian was presented and was accepted, ordered
printed and referred for audit to a committee consisting of Professor Grif-
fin and any other Pittsburg members whom he might select.

The Treasurer’s report for the years 1916 and 1917 was accepted and
referred to an auditing committee consisting of Drs. Latham and McCalla
of Chicago.

The Society approved the recommendation of the Treasurer that the
fiscal year be regarded as extending from Dec. 1 to Nov. 30.

The following officers were duly nominated and elected for the con-
stitutional periods: President, Professor L. E. Griffin, University of Pitts-
burg; First Vice-President, Dr. H. M. Whelpley, St. Louis; Second Vice-
President, Professor C. O. Esterly, Occidental College; Secretary, T. W.
Galloway, Beloit College (for two years); Custodian, Magnus Pflaum, Esq.

In connection with the re-election of Mr. Pflaum it was noted that he
has been custodian for eighteen years continuously since the formation of
the office, having been Treasurer for three years before. A cordial vote
of appreciation was extended him for this long and efficient service.

Professor Max M. Ellis of University of Colorado and Professor
J. E. Ackert of Kansas State Agricultural College were chosen as the elective
members of the Executive Committee for 1918.

Professors Grifin and Galloway were appointed a committee to approve
and print the minutes. Adjourned.

T. W. Gatitoway, Secretary.

72 MINUTES

SPENCER-TOLLES FUND
Custodian’s Report for the Years 1916 and 1917

Amount reported in TG soi cide eee oe eee $4489.32

Sane Oly 1916) Dividends ee ioe Tay aia $ 134.67

ely) 45 1SIG: SDivademasnite s). ade sais tet rordina we ties Miele eee 138.69

Pec.) 6, 1916: (Salei pti ransactions :))s ))s/.6 bonis eaaicexeene 60.00

Bec:/15, 1916) Sale jor Mransactions 3.024 issn fee emote 60.00

CU ST TOG. So vaAeTES Hs) lee 6 Sys cis Wis ccioere ulead wivwele etree 142.86 536.22
$5025.54

etl ds OL7 A ONVIGEMUS Cen RU Wl tno menhe Gesu a ralg $ 150.75

PCOS HADET. EMVIGEMES ) le odie ois Netemieiniojctetale eee cetes aieereee 155.28 306.03
$5331.57

GRAND TOTALS

AU CONETIBUHORS ae ite Nieisd Sa uitlo miaalie eis a eee a dwrmcaiereere $ 800.27

ATE Sales Of TransSactecarsy ye ee Ne te ate le 878.38

ATE Te KMEmbEnSHigs eee snot s cet ole ole eemretel aie tele lee 300.00

AMD Taterest and (Dividends' (253.0. sess assay ein oe ecient 3542.92 $5521.57

LESS

PAU MaraMIPS eee. iota el wa mbliclen teldian vad cheisiets eke ale muntomtetetm mete $ 150.00

WlbyDites on) tafe ‘Members tick. ocias oc ce sae cae cule wits 40.00 190.00
$5331.57

Life Members: (Robert Brown, dec’d); J. Stanford Brown; Seth
Bunker Capp; Henry B. Duncanson; A. H. Elliott; John Hately.
Contributors of $50 and over: John Aspinwall; Iron City Microscop-
ical Society; Magnus Pflaum; Troy Scientific Society.
Macnus Prraum, Custodian.
Pittsburgh, Pa., Dec. 29, 1917.

We, the undersigned committee, hereby certify that we have carefully
examined the above account for the years 1916 and 1917 and found the same

correct.
L. E. Grirrin, Chm. Auditing Committee.

MINUTES : 73

ANNUAL REPORT OF THE TREASURER OF THE AMERICAN
MICROSCOPICAL SOCIETY

December 24, 1915, to December 24, 1916

RECEIPTS

Bates on, band)tromy TODS i oe Ae aU crt) er et $ 476.65
Merabershigi QUES: | sos oe s.2 ai ce Re AEROS Pe AAR EEE Ur 532.10
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STI SC DCL Space erat Feat tene aS RUA a Epa a lhe as ayreria an equ uN Enh Sma a 302.20
Salesvo fee DEATISACHONS a Va seer alee ical eae chat ieraih cpa nia atsaees Sieitepall 150.40
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MEAGRE nei S ok Aha Sata eer ra GTA «ele chaser erate: Se tahets hoa 2.68
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EXPENDITURES
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Office expenses; stenography, supplies, etc.—

SSE CE CEE Wine sch eters ta coc Sica teas aCe aoe = are eC tS ce ee eee era 134.59

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Postage and express—

CS ECSUTLE 9 5 pr NRE MERE oe UL A ea UR NC Pr REP BT 2 SR St ah 109.78

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Ra ated E ALISACTIONS) iaig(s nosed at en ne eae ee eae he pee 16.16
Spencer-Tolles Fund from sales of sets of Transactions to U. of Pa.

ANGTEOS NOEs Dames Ue oe aceaaan rd beth st UL e anes Sethe mS 120.00
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Expenses of Secretary at Columbus meeting, 1915................... 31.65
SVEST EAA Be eh NPE RY iP lW SRA aS e a LEN ACEO Och ALOR A 5.70
woe. Eheaes Tae tell 231 imate PB ante SR ih Cpe AA ce CAN PO Rae SE 273.71

$1777.93

Respectfully submitted,
H. J. Van CLeAvE, Treasurer.
Feb. 15, 1918.
We hereby certify this statement corresponds with the Treasurer’s book.

V. A. LatHaM,
GEORGE Epwarp FELL,
Auditing Committee.

74 MINUTES

ANNUAL REPORT OF THE TREASURER OF THE AMERICAN
MICROSCOPICAL SOCIETY

For the year beginning Dec. 25, 1916, and ending Dec. 21, 1917

RECEIPTS
Balance on hand ‘from 1916. 0/0/20. Sarees at eee eee ee $ 273.71
Membership iGUesie rari sie nate vss es ele Ae ANCL a ss Seed at a 880.00
MIEIATION s TEES it artite ct Cates isisle eos oes yar e e cle a means Caan ane a ee 42.00
BSISGTIDELS OAR hee ep con ake dice Sic shatters tie Sieieiu oho Me wld eine Ea aa 119.80
MIIGUOE SL TARSACTIONIS. ollctslosciatass ito Bele de ak hhc coe elas et ae 174.00
DROLET RISET SNe Casey eh RCo gece See tl Ul UR tel a 435.00
Partial mavinentutor Cuts, 2. ccc cee sand) ion Sabe vei cas cine oa ee eee 23.14

MRIEAN CVERENPES ei eitcin es otan Galstad eels bie wrens eater blo son gt ne $1947.65

EXPENDITURES

Printing) transactions. -voOlime (5. t10.04 s mntoesa sae aes aml oe eee $ 259.28
Prntine Transactions, volume 36, nos; 1,:2,'and 3 2)..4.....0 Gaon 543.74
Pistes) tor ylransactions volime roo slOn adore ceiciae eis cists sitters ieee 29.64
Phites) tor) Transactions, volunie 36, nos. 1) 2) 3; 49.5... =. se eee 78.35
Postace arid iepressy ak ole iss etic cheeks okeniss Wid ns oe teen ae ree 75.42
Office expenses—

SS ECTELAEY |)’ aes c chon araie ecb gS wide e ocala oleh do oun sn ee eR ance atin aie ee eno 73.37

PTCASHEET ee veal Othe eek iced wi ttemaeeinilove aie GEC ene serch e ieee ea 32.26
In part payment of Secretary’s expenses to Pittsburg meeting...... 50.00
Binding set’ of; 0 ransactions fOr Sale’. cic eicisie eters sole wists) ng <u os 20.00
FISECIIAREOUS ITEMS s,s Wioislece ewddie va bid nes detec ca ies Oe ee 16.02
Babatice-on Hands: <.sceg Sh eeeioseanhccead eee oe SRG ata ee 770.57

$1947.65

Respectfully submitted,

H. J. Van CLeAvE, Treasurer.
Feby. 15, 1918.

We hereby certify this statement corresponds with the Treasurer’s book.
V. A. LatHam,
GEORGE EDWARD FELL,
Auditing Committee.
Feb. 12th, 1918.

I hereby certify that I have examined the vouchers and accounts of
H. J. Van Cleave, Treasurer American Microscopical Society, for the period
ending Dec. 21, 1917, and I find the balance of $770.57 as shown on the books

to be correct.
F. S. Jonnson, Public Accountant.

TRANSACTIONS

OF THE

American
Microscopical Society

ORGANIZED 1878 INCORPORATED 1891

PUBLISHED QUARTERLY

BY THE SOCIETY

EDITED BY THE SECRETARY
T. W. GALLOWAY

BELOIT, WISCONSIN

VOLUME XXXVII
NUMBER Two

Entered as Second-class Matter December 12, 1910, at the Post-office at Menasha,
Wisconsin, under act of March 3, 1879.

The Culleniate Press
GrorcE BANTA PUBLISHING COMPANY
MeENASHA, WISCONSIN

OFFICERS

POSIT Teihe, VAST D AN Ca a 0. Bene aa ea eae Pee orisha ee Ch as LIEN Pittsburg, Pa.

naesiavoce: President: EH. Nt. WHELPLEY. 3.2...0000. oe Se St. Louis, Mo.
eam Vice-president: ©. QO: WSTERTY: 20: os eee en ee Los Angeles, Cal.
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ELECTIVE MEMBERS OF THE EXECUTIVE COMMITTEE

Us Ie WS CS ot eR ae eee tC N Eke te) CM ROR AG by (Aan eRes ROR Rem Pee ae wr 8 Boulder, Colo.
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EX-OFFICIO MEMBERS OF THE EXECUTIVE COMMITTEE
Past Presidents Still Retaining Membership in the Society

ALBERT McCALLA, PhD., F.R.M.S., of Chicago, IIl.,
at Chicago, Ill., 1883
Gero. E. FELL, M.D., F.R.M.S., of Buffalo, N. Y.,
at Detroit, Mich., 1890
Smion Henry Gace, B.S., of Ithaca, N.Y.,
at Ithaca, N. Y., 1895 and 1906
A. CLIFFORD MERcER, M.D., F.R.M.S., of Syracuse, N. Y.,
at Pittsburg, Pa., 1896
A. M. Biere, M.D., of Columbus, Ohio,
at New York City, 1900
C. H. E1GENMANN, Ph.D., of Bloomington, Ind.,
at Denver, Colo., 1901
E. A. Birce, LL.D., of Madison, Wis.,
at Winona Lake, Ind., 1903
Hnery B. Warp, A.M., Ph.D., of Urbana, IIL,
at Sandusky, Ohio, 1905
HERBERT Oszorn, M.S., of Columbus, Ohio,
at Minneapolis, Minn., 1910
A. E. HErTz1ER, M.D., of Kansas City, Mo.,
at Washington, D. C., 1911
F. D. Heatp, Ph.D., of Pullman, Wash.
at Cleveland, Ohio, 1912
CHARLES BROOKOVER, Ph.D., of Louisville, Ky.,
at Philadelphia, Pa., 1914
Cuartes A. Koron, Ph.D., of Berkeley, Calif.,
at Columbus, Ohio, 1915
M. F. Guyer, Ph.D., of Madison, Wis.,
at Pittsburg, Pa., 1917

The Society does not hold itself responsible for the opinions expressed
by members in its published Transactions unless endorsed by special vote.

TABLE OF CONTENTS
For VoLuME XXXVII, NuMBER 2, Aprit 1918

Three New Species of Amebas, with Plates, by Asa A. Schaeffer
Spermatogenesis of the Dog, with Plate, by Julian Y. Malone.......................
Thigmotactic Reactions of the Fresh Water Turbellarian, Phagocata Gracilis,
Leidy, by Bernol 4. "Weimer .--0i5.:52.2..0.-0. oe ote ee
Additions to Our Knowledge of Unionicola Aculeata (Koenike), with Plate, by
1 OPA GA Or: | Rem nS A Dr gtd e. YG Pn Sera nel ae Gat Mlb hrs i Bocce
Notes and Reviews: Methods for Studying Living Trematodes, William Walter
Cort; A Substitute for Euparal, E. S. Shepherd; Chromosomes of Ranatra
Sp?, A. M. Chickering; Notes on Collecting and Mounting Rotifers; Methods
of Preserving Certain Marine Biological Specimens; The Silverman Ilum-
MmAtor Lor WVECOSCOPES: -.<oiees cots osacsens sees cota e reese eee ot cnc ses ec ea nae ee a

125

TRANSACTIONS

OF
American Microscopical Society

(Published in Quarterly Instalments)

Vol. XXXVII APRIL, 1918 No. 2

THREE NEW SPECIES OF AMEBAS: AMOEBA BIGEMMA
NOV. SPEC., PELOMYXA LENTISSIMA NOV. SPEC.
AND P. SCHIEDTI NOV. SPEC.

Asa. A. SCHAEFFER

The classification of the amebas' is peculiar in that two methods of
species determination are followed. The larger amebas are classified
according to the characteristics possessed by the cytoplasm and the
general character of the vegetative stage, while the smaller amebas
are being described according to the method of nuclear division pre-
vailing during reproduction. In the larger amebas a study of nuclear
division is extremely difficult and for many species impossible, owing
for one thing to our ignorance of culture methods by means of which
these species could be raised in abundance. On the other hand the small
amebas exhibit so little cytoplasmic differentiation that specific deter-
minations on this basis seem impossible.

A specific determination is interesting, however, from at least two
points of view. One is the viewpoint of establishing blood relationships
of descent between the different species, or systematics proper. The
other point of view is a purely practical one, i.e., quick identification.
The physiologist or the experimentalist wants a quick and correct method
for identifying the organism he is working with. It is obvious therefore
that if an ameba possesses characteristic cytoplasmic differentiations
which may be observed at any time, the ameba will come to be recognized
by these characteristics rather than by a complicated series of nuclear
events which occur only occasionally and are frequently made out only
with difficulty. In short, as a means of identification, cytoplasmic char-

1 The word ameba is used as a common name for naked rhizopods lacking internal

skeletons such as Amoeba, Pelomyxa, Protamocba, Endamoeba, Négleria, Hyalodiscus,
Dinamoeba, Vahlkampfia, etc.

80 A. A. SCHAEFFER

acters are preferable where they exist; where these do not exist, recourse
may be had to the nuclear division process.

All the larger amebas possess cytoplasmic differentiations in sufficient
number and conspicuousness to serve as a ready means of recognition.
Many of these characters are subject to very slight variation as a result
of changes in the environment. Individual isolation pedigrees carried
on for upwards of a hundred linear generations together with many
collateral lines under varying food conditions, showed that most of the
cytoplasmic characters are hereditary and practically uninfluenced by
what might be said to be the most common environmental changes
(Schaeffer, Science, 1916, p. 468). Amebas are therefore in this respect
like all other groups of animals and the method of classifying them ac-
cording to cytoplasmic differentiations is therefore sound.

These considerations should convince especially our younger micro-
scopists that the investigation of our larger amebas is not nearly as diffi-
cult or forbidding a field as might be imagined from the great amount
of labor that has been expended on the study of the life histories and
nuclear phenomena of some soil and parasitic amebas during the past
decade. The fifty or so species of aquatic amebas thus far described
represent beyond any question only a very small fraction of the number
of species in existence, and this number of known species could probably
be doubled within a few years by careful examination of our marshes
and ponds.

AMOEBA BIGEMMA NOV. SPEC.

Diagnosis. Size in locomotion, 100 to 300 microns long. Form very changeable.
Pseudopods, numerous, tapering, blunt, never with sharp points. Surface smooth,
no fine folds or ridges. Endoplasm usually containing numerous small twin crystals;
crystals attached to ‘excretion spheres.’ Movement rapid, about 125 microns per
minute. Nucleus single, spherical or slightly ovoid, about 12 microns in diameter;
chromatin in small masses clumped loosely together in the center of the nucleus in a
nearly spherical mass about 6.5 microns in diameter. Contractile vacuoles small
about 15 microns in diameter; numerous; no coalescence among them; systole slow.
Endoplasm filled with small vacuoles. Food: flagellates, ciliates, diatoms, rhizopods,
nematodes, vegetal tissue, etc.

This ameba, for which the specific name bigemma is proposed, re-
sembles to some extent the figures and descriptions of Parona’s digitata,
Mereschkowsky’s angulata, Gruber’s spumosa and Penard’s ves pertilio.
In fact I regarded it at first as the angulata of Mereschkowsky or the
ves pertilio of Penard, which it occurred to me might possibly be synony-

THREE NEW SPECIES OF AMEBAS 81

mous. But after further study of the characters of this ameba I began
to suspect that my earlier conclusions regarding its specific reference
might be mistaken. I accordingly investigated the specific characters
of this ameba in connection with some experimental work, under widely
varying conditions for about three years, and compared my observations
with the published reports of earlier investigators of amebas with the
result that I am unable to confirm the specific descriptions by any of the
authors named from this ameba.

In the first place, Mereschkowsky’s (’79) description is extremely vague
(pp. 203-204) Mereschkowsky says the plasm of angulata is trans-
parent, that it contains two kinds of grains: a few large ones and numer-
ous small ones. About three contractile vacuoles and a small round
nucleus are present. Few, ‘“‘am ende zugespitzten (doch nicht wie bei
A. filifera mit welcher A. angulata viel Aehnlichkeit hat) und die gestalt
dicker, breiter Kegel habenden, vom K6érper ausgehenden Pseudopodien
characteristisch. diam. 0.0235.” Movements very rapid. The figure
illustrating this description is very crude. With the exception of size,
this description as far as it goes might apply to a number of species of
amebas. The size, 23.5 microns, is very much smaller than that of
bigemma.

Parona’s description of A. digitata (1883. Essai d’une Protistologie
de la Sardaigne. Arch. des Science physiques et naturelles. T. 10. Troi-
siéme periode. p. 225-243. 1 plate) is somewhat more definite than Mer-
eschkowsky’s of angulata. A. digitata possesses a very granular endo-
plasm, a rounded and conspicuous nucleus, a large contractile vacuole,
““pseudopods longs, conique et aigus,’’ pseudopods always in small num-
ber. Movement is rather slow. Size, 63 microns (p. 228). The only
three characters which may be considered distinctive are the size, the
conical and pointed (the figure shows needle points) pseudopods, and
the number of contractile vacuoles. None of these characters however
are found in digemma. Parona makes no mention of vacuoles in the
endoplasm, which, if he had seen a bigemma, he could not have helped
seeing, since these vacuoles are quite as conspicuous as the nucleus.
There can be little question, I think, that Parona described another
ameba than bigemma under the name digitata.

Leidy(79) figures several amebas resembling bigemma, vespertilio
and digitata more or less closely, but he regarded them all as varieties
of proteus, or as forms of uncertain specific reference.

82 A. A, SCHAEFFER

In his description of A. spumosa Gruber is no more explicit than
Mereschkowsky in the instance mentioned. Spumosa has broad flat
pseudopods, a vesicular nucleus, an endoplasm filled with vacuoles, no
granules, a size of 25 microns, according to Gruber. As emended by
Penard (’02) spumosa possesses these characteristics: A length of from
fifty to one hundred and twenty-five microns; form resembling the foot
of a goose, with very fine longitudinal lines on the surface; numerous
vacuoles; contractile vacuole as much as thirty microns in diameter;
a great many bicuspid granules of very small size in the endoplasm;
nucleus deformable like that of A. limax; a compact nucleolus with a
narrow margin of nuclear sap between it and the nuclear membrane.
Althought I am inclined to accept the emendation of Penard because of
its making for greater definiteness and stability in this difficult genus,
yet it appears to me that instead of really emending or elaborating
Gruber’s description, he actually describes a new and different species.
It is evident that another ameba than bigemma was under observation
by both Gruber and Penard when these authors wrote their descriptions
of A. spumosa.

Penard’s description of A. verspertilio (1902, Faune Rhizopodique du
bassin du Leman. Geneva, pp. 714) is as follows: size, about seventy
microns length; pseudopods have always a conical form, their extremities
being usually sharp pointed although the point may be slightly rounded
occasionally for a moment’; posterior end sticky, dragging debris along
as it moves forward; a profusion of extremely small green grains, and
sometimes large excretion grains, in the endoplasm; a sperical nucleus
with a compact nucleolus which is often covered with fine points; con-
tractile vesicles one, two or three; almost always a considerable number
of vacuoles appearing and disappearing as if they were contractile in
the endoplasm (pp. 92-95). Penard’s figures (p. 94) resemble in a
general way the figures of bigemma, but in the important points such as
the number and character of vacuoles, the shape of the pseudopod
extremities, the relative diameters of the nuclear membrane and the
nucleus, the character of the “nucleolus,” stickiness of the posterior
end, inclusions, etc., there is little resemblance between ves pertilio
and bigemma.

2. . . les pseudopodes ont toujours une forme conique, anguleuse; leur extermite

est en principe acérée; mais parfois la pointe peut s’arrondir pour un instant (p. 93).
His figures all show sharp needle points on the extremities of the pseudopods.

THREE NEW SPECIES OF AMEBAS 83

The Amoeba bigemma is of medium size, being usually from 100 to 300
microns long in locomotion. Occasionally the size is very much greater.
In several old cultures the amebas frequently arrived at a length of 500
microns in locomotion. As a rule, the average size in new cultures is
about 150 microns.

10:37 10:373

10:38% FA aS = a 8 ‘
/0:394 ESS 40:38Z = 0:39%

Fig. 1. Camera lucida drawing of an A. bigemma in locomotion during the process
of fission. The figures indicate the time in hours and minutes. The drawings from
10:3714 on were moved up from their true position the length of the vertical line below;
likewise for all drawings from 10:36 on. Note slowing down in the rate of locomotion
as the fission crisis was approached, and the gradual increase of speed after division.

Although the general shape of this ameba is subject to very great
variation, yet the various transformations are very characteristic. Per-
haps the most characteristic feature of these transformations is the
tongue like pseudopods, usually short, sometimes very long, which are
continually being thrown out at the anterior end and on the free surface
during locomotion (PI. VII, fig. 2). These projections are frequently more
or less conical, though more often perhaps they have somewhat the shape

84 A. A. SCHAEFFER

of a hart’s tongue in outline. These projections are not pseudopods in
the same sense that the projections in dubia or in proteus are pseudopods.
In the latter species a pseudopod gives direction to locomotion, that is,
the whole ameba frequently flows into a pseudopod. In bigemma, how-
ever, the whole ameba almost never, if at all, flows into a pseudopod;
but the tips of the pseudopods advance at about the same rate of speed
as the anterior end of the ameba as a whole advances. The pseudopods
are, therefore, to be regarded rather as “toes” on the single pseudopod
of which this ameba usually consists. During active locomotion the
general shape of the body is most frequently triangular, with the broad
base advancing, and more or less compressed. When the animal is dis-
turbed it assumes a spherical shape. When suspended in the water for
some time the ameba assumes a shape closely resembling that of the
rayed state of a radiosa. From a central spherical mass of about thirty
or forty microns diameter, long, slender, tapering pseudopods are thrown
out which are of a much more permanent character than the pseudopods
thrown out during locomotion. These projections are sometimes per-
fectly straight and of equal size and disposed opposite to each other on
the spherical mass. More often however they are curved and of irregu-
lar shape and size. Their number is usually from six to eight. All the
pseudopods formed during locomotion or while suspended are blunt,
very definitely blunt. Of all the thousands of individuals which I have
examined in all the different stages and cultural conditions, I have never
seen any but definitely obtuse pseudopods. The photographs of live
amebas, figs. 4 and 5 indicate the degree of obtuseness characteristic of
the pseudopods of this species (Pl. VII).

The streaming of protoplasm during locomotion presents some points
of interest. One does not observe the endoplasm flowing slowly in a
definite direction in this species as one may in proteus or dubia, for
example, but the streaming is jerky and irregular. The endoplasm
seems to drain from the sides and posterior end toward the anterior end
against numerous obstructions which often give way. Thus there are
developed momentary counter or cross currents and eddies. The peculiar
character of the streaming is due to several causes. In the first place
the numerous pseudopods are formed and retracted without definite
reference to the direction of locomotion. Then again the upper surface
of the ameba is not level but extremely irregular; one observes a con-
fusion of high ridges and deep depressions thrown together without

THREE NEW SPECIES OF AMEBAS 85

observable order. From the depressions more or less permanent pillars
of stiffened ectoplasm pass down to the lower surface obstructing the
flow of endoplasm. When these pillars give way, as they frequently
do, the bottom of the depression is suddenly pushed upward as if the
endoplasm were under considerable internal pressure. Another cause
of the irregularity of endoplasmic streaming is due to the fact that the
anterior end does not advance steadily over the whole front, but by a
series of waves here and there. Such a wave consists usually of a web
of ectoplasm flowing out between and connecting two or more of the
pseudopods; then the web halts momentarily while the pseudopods push
out again or while new pseudopods form.

The rear end of the ameba usually plays little part in locomotion in
a special way; usually the rear end is smooth and free from any pseudo-
podial projections. Occasionally, however, a long thin flat pseudopod
may be rapidly thrown out near the posterior end which fastens itself
to the substratum so well that considerable force is required to dislodge
it (fig. 2). The ameba sometimes becomes stretched out to several times
its usual length before the pseudopod is pulled loose. This behavior
also results often in another phase of movement that I have much more
frequently observed in this ameba than in any other, viz., some of the
endoplasm begins to flow toward the posterior end until the ameba is
cut almost in two in the middle. The connection is so thin that one
looks every second for the connecting strand to break, but it does not
break. Sooner or later one or the other portion of the endoplasm flows
back and the whole mass again becomes unified in streaming.

The rate of movement is rapid, being about 125 microns a minute.
Although the anterior end advances very unevenly and irregularly, there
are, nevertheless, long segments in the path of an ameba moving on a
plane surface that are straight. There is present in this ameba, there-
fore, the same tendency to keep on moving in the direction in which it
started to move as there is in proteus (Schaeffer, ’12, ’18; this point will
also be discussed at length in a paper soon to be published).

There is present in this ameba a layer of very thin protoplasm on
the outside of the ectoplasm, as usually defined, which moves forward
over the ameba in the same direction as that in which the ameba moves,
but at a more rapid rate. This is clearly shown by the forward move-
ment over the ameba of small particles which cling to this layer of pro-
toplasm (Schaeffer, ’17).

86 A. A. SCHAEFFER

During locomotion there is usually a broad zone of clear ectoplasm
at the anterior end. Not only are the smaller pseudopods in this region
free from granules but the intervening spaces also consist of ectoplasm.

The nucleus is easily seen. Unless obscured by food masses photo-
micrographs of the living amebas usually show it (fig. 4.). It is spherical
or very slightly ovoid (fig. 3). In an ameba of about 200 microns length
the nucleus is about twelve microns in diameter. The chromatin con-
sists of very small ovoid granules collected together in the centre of
the nucleus in a slightly irregular oval mass of about six or seven microns
diameter. The color of the chromatin is a pale bluish yellow green.
Between the chromatin mass and the nuclear membrane, which is per-
fectly transparent, there is a zone of clear nuclear sap. The nucleus is
single, though occasionally an ameba is found with two nuclei, which
statement is true of course for practically all amebas. The nucieus is
deformable though it is not often that one observes striking deformations
as it is swept along by the endoplasm.

In one culture of iarge amebas of this species the nuclei were about
twenty-eight microns in diameter, and the chromatin mass of irregularly
spherical shape was about fourteen microns in diameter. When these
amebas were slightly squeezed under the cover glass one or two masses
of mostly perfectly homogeneous pale bluish yellow green material was
pressed out from the chromatin mass. There were present here and there
large spheres of denser material of the same color as the homogeneous
masses. Both the granular mass and the homogeneous masses rounded
themselves up and collected near the centre of the nucleus. The material
making up the masses seemed to be of the same sort, though I did not
employ staining methods to determine whether the interior of the gran-
ular chromatin mass was really chromatin or some other substance.
It is, however, interesting to know that at least some of the material
inside the chromatin mass is not granular while the outside material
is.

There seem to be three kinds of vacuoles in this ameba in so far as
their functions are concerned. The endoplasm contains scattered about
in it everywhere numerous (100 or more) small clear vacuoles of various
sizes, mostly under ten microns in diameter, which may be called per-
manent vacuoles. What the function of these vacuoles is, or what con-
ditions are necessary to their origin, remains unknown. That all of
these may become contractile is hardly possible, unless they retain their
identity in the ameba’s body for many hours.

THREE NEW SPECIES OF AMEBAS 87

The second kind of vacuoles are the contractile vacuoles. These have
‘the same general appearance as the permanent vacuoles, excepting that

they are more refractive to light. These vacuoles arrive at a diameter
of about fifteen microns before they contract. It is very seldom that
a larger size is attained. The diastole of these vacuoles is rather slow.
The systole is also very slow, occupying from two to six seconds. There
are a number of contractile vacuoles present at one time. Under favor-
able conditions as many as four may be observed to be in the process
of diastole at one time. The general appearance of the later stages of
a contractile vacuole, that is, higher refractive index of its contents,
possibly indicates that these vacuoles are different from the permanent
vacuoles from their beginning.

The third kind of vacuole, which may be called the fecal vacuoles,
are not frequently met with in amebas. These are large spherical vacu-
oles containing in proportion to their size a very small amount of fecal
matter. These may reach a diameter of twenty or twenty-five microns
and in occasional large specimens a diameter of from forty to sixty
microns. I have not been able to ascertain whether the vacuole ori-
ginates around the fecal matter or whether after the vacuole is partly
formed the fecal matter is voided into it. Since however, the vacuole
usually becomes very large, it is evident that the later stages of the
increase in size is due to the contained solid matter. The systole of these
vacuoles is very slow. The liquid contents is first expelled and then
after a pause the solid matter is thrown out in the way common to amebas
generally.

None of these different kinds of vacuoles seem to grow by coales-
cence; at least from extended observation with this point in view I have
never seen a single case of coalescence, although vacuoles frequently
remain in close contact for a minute or longer.

Another very interesting element in the endoplasm is the crystals.
These are usually very numerous and conspicuous, ranging in size from
one and one-half to two and one-half microns in length. The general
shape is like that of an hour glass or dumb-bell. They seem to be formed
of two bicuspid crystals attached to each other by their apices (figs.
2,3). Under ordinary light they appear dark gray in color; but in po-
larized light they show up very brightly, and then their twin structure
becomes very evident. This is the only ameba known in which such
a twin structure of crystal formation isfound. The polariscope shows an

88 A. A. SCHAEFFER

unmistakable twin structure, however, in only about half the cultures
I have so far investigated. In the others the two points of light are
joined by a bar of light so that one sees a band of light not constricted
in the middle. It may be inferred, however, that in these cultures the
earlier stages of crystal formation are also on the twin pattern. It has
been found that the character as well as the amount of food influences
the size and to some extent the character of the crystals formed. Per-
haps the most striking thing about the crystals in this ameba is the fact
that they are always attached to the so-called excretion spheres when
these are present, as they nearly always are (fig. 3). There is never
but one crystal attached to a sphere. The size of the sphere bears no
relation to the size of the crystal, the spheres being in some cases just
barely visible, while the crystals may be two microns long. When the
the spheres are small the crystals are always attached to them at their
middle. The sphere and crystal bear a remarkable resemblance to a
fish embryo lying on its egg yolk, and they form interesting objects
for observation as they tumble along in the streaming endoplasm. Occa-
sional twin crystals are observed apparently free from attachment to
spheres, but it is possible that the spheres are extremely minute in such
cases, too small to be seen. The excretion spheres rarely exceed a dia-
meter of three microns. In some cultures a few bicuspid crystals with
irregular sides are observed occurring singly. The maximum size of
these is about two and one-half microns. Occasionally also two twin
crystals are found crossed, (fig. 3). Altogether, the crystals form the
most definite specific character of this ameba, and the presence of such
crystals attached to spheres in an ameba may be regarded as definitely
proving its specific identity.

This species is a voracious feeder. Flagellates, shelled rhizopods
ciliates, rotifers, nematodes, diatoms, etc., and especially bits of vegetal
tissues and masses of bacteria, are readily devoured. The body is fre-
quently stuffed with food.

This ameba is one of the hardiest known to me. I have kept numer-
ous and continuous cultures, after being well established, for several
years without much difficulty. The species is subject to very little
variation excepting size. In nature this species must be considered
rare, though it is found frequently where large masses of vegetation are
undergoing decay in quiet water.

THREE NEW SPECIES OF AMEBAS 89

PELOMYXA LENTISSIMA NOV. SPEC.

Diagnosis. Length in locomotion, 75 to 125 microns. Body usually very much
compressed and applied closely to substratum. Changeable in shape, general out-
line oval with few pseudopods. Quiescent stage with numerous pseudopods of various
shapes. Color of body brownish; of protoplasm, pale bluish. Uroid of fine or large
root like projections. Rate of movement very slow, from 1 to 2 microns per minute.
Nucleus spherical, about 14 microns in diameter. Chromatin in a spherical layer of
granules about 11 microns in diameter, with spherical body about 2.5 microns in the
centre of the nucleus. Two nuclei frequently present. Contractile vacuoles numerous,
40 or more; maximum size about 10 microns; systole sudden; diastole very slow. Num-
erous or few small irregular crystalline masses present. Numerous bacterial rods of
about 4 microns length present. Only very few refractive (starch) bodies present.

This pelomyxa is readily recognized by its small size and its very
slow rate of locomotion (PI. VIII, fig. 6). It is, in fact, much the slowest
moving pelomyxa thus far reported, and I, therefore, propose the specific
name of /entissima for this species.

I have found this organism in large numbers in old cultures from the
marshes of Lonsdale on several occasions. But on account of its small
size and its habit of flattening itself out on and sticking close to the sur-
face on which it moves, it more readily escapes detection than other
amebas of the same size. The color of this species is a dull brownish,
somewhat like that of P. belevskii, but not so deep a shade, owing to its
smaller size. This color is, of course, due to the contained food materials
and the indigestible remains of food objects. It seems to be a habit of
this and other pelomyxas to carry for a long time indigestible materials
from food objects before excreting them, if indeed some of this material
is ever excreted while the animal is in the vegetative stage. The color
of the protoplasm is of a bluish violet tint.

This pelomyxa as distinguished from the other species, flattens itself
out and sticks very close to the surface during locomotion. At such a
time it is thickest in the centre and gradually becomes thinner as the
periphery is approached. Around the entire animal there is a clear zone
of protoplasm which is hyaline and very thin and of which the exact
outer limit is very difficult to see. Pseudopods are continually being
extended and retracted from the entire periphery of the animal except
from the posteriorend. These pseudopods are of clear protoplasm except
for a small number of very pale bluish granules which are frequently
found at their bases. The pseudopods are broad or narrow and always
blunt. They do not usually determine the direction of locomotion.

90 A. A. SCHAEFFER

The posterior end terminates in a uroid or group of root-like pseudopodial
projections attaching the organism to the substrate (fig. 6). They play
a part in locomotion but just what their function is has not been carefully
determined. It is certain, however, that the uroid is not necessary to
locomotion to the same extent as in P. schiedti.

Locomotion in this form is so extremely slow that it is difficult to

tell just how it is accomplished. Movement occurs at the rate of from
one to two microns a minute, so that it takes from 30 minutes to an hour
and a half to move the distance of its own length, or three and one-half
months to creep around a baseball. There is a slight but continual and
irregular movement of the endoplasm of an oscillatory sort, which,
together with slight changes of body shape and the retraction and pro-
jection of pseudopods, masks the definite forward movement of the
pelomyxa. Figure 9 shows that this movement undoubtedly exists.
But under ordinary circumstances it is impossible to detect definite
and continuous forward streaming of the endoplasm. It seems as if
forward movement was the sum of all the separate local streamings.

Fig. 9. Camera lucida sketch of a moving P. lentissima to show rate of, and form
changes during, locomotion.

When suspended in the water or when loosened from the substratum
this ameba is remarkable for the number of pseudopods which it throws
out from all sides (fig. 7). This habit may indeed be regarded as a
distinguishing characteristic of this species while in this stage. The
pseudopods are usually more or less conical in shape though they always
have blunt ends. The majority are of simple form, others are branched,

Se eee

THREE NEW SPECIES OF AMEBAS 91

while some are of very odd shapes. Their length is variable, the maxi-
mum being about 50 microns. As might be expected from their rate of
locomotion, the transition from a spherical shape in the suspended stage
to the locomotive stage consumes much time. As the organism again
begins to move, a broad wave of clear ectoplasm appears at some point
near the substrate. The wave slowly enlarges as the pseudopods are
withdrawn, and gradually the locomotive form reappears.

The nucleus of this species presents several points of interest. In the
first place there are usually two. In perhaps three out of four indivi-
duals examined two nuclei were found. In no case did I see more than
two. It seems that in this species it is the normal condition for a con-
siderable interval of time to supervene between division of the nucleus
and the division of the organism. The two processes do not seem to be
directly dependent on each other. In the case of most other amebas cell
division must closely follow nuclear division, or else in the majority
of cases it will become impossible for cell division to take place and the
animal in consequence dies (Schaeffer, ’16). So that what is normal
in division sequences for Pelomyxa lentissima is pathologic for Amoeba
proteus, for example. A P. lentissima with two nuclei may be looked
upon as an individual whose cytoplasm has not yet divided.

Another point of interest with regard to the nucleus of this species
is that a central body is clearly observable in the living condition. The
composition of this body has not been investigated. The diameter of
this body is from two to two and one-half microns. Its appearance is
similar to that of the chromatin granules.

The general appearance of the nucleus is somewhat like that of P.
belevskii. It is spherical, about fourteen microns in diameter, and con-
tains a spherical layer of chromatin granules of about eleven microns
diameter. Not very much can be said about the physical character of
the nuclear membrane owing to the very slight movement of the endo-
plasm. The nuclei are usually found somewhere near the centre of the
animal.

The contractile vacuoles are numerous. In one individual there
were at least sixty, but whether all were contractile or not could not be
determined. The average maximum size of these contractile vacuoles is
about ten microns, though many contract when only five microns or so
in diameter. Occasionally one may reach fifteen microns before con-
traction. Consonant with the slow rate of locomotion is that of enlarge-

92 A. A, SCHAEFFER

ment of the vacuoles. A vacuole of five or eight microns diameter may
take fifteen minutes or longer to enlarge to ten microns, followed by
contraction. The systole is, however, relatively rapid, occurring usually
in about one second.

The spectroscope shows some very irregular small crystalline masses
in the endoplasm sometimes numerous, usually comparatively few. Of
“refractive bodies” (starch grains) there are only a few small ones
present. Other inclusions in the ectoplasm are the bacterial rods, dis-
tinctive of the genus, reaching a length usually of four microns, very
rarely of eleven microns; and the very numerous brownish masses of all
sizes and shapes so common in several species of pelomyxas. In addi-
tion to these bodies there are also numerous very small greenish blue
granules found in the endoplasm.

Besides an occasional diatom or shelled rhizopod, I have found no
recognizable food bodies in this species.

PELOMYXA SCHIEDTI NOV. SPEC.

Diagnosis. Length in locomotion about 75 microns. Usual shape ovoidal. Color,
brownish olive green, almost opaque. ~Pseudopods very rarely formed. Protoplasm
fluid. Movement by eruptive waves of endoplasm partly reflected back along the
sides. Rate of movement about 95 microns per minute. Nucleus sometimes single,
usually double; spherical, about 7 microns in diameter. Chromatin granules in
the form of a hollow sphere immediately under the nuclear membrane or at a slight
distance from it. Contractile vacuoles small, numerous; maximum size about 4
microns; diastole rapid; systole instantaneous. Starch grains very numerous, irregular
in shape, olive green in color, maximum size about 6 microns. Numerous bacterial
rods present of 3 to 4 microns length. Small uroid always present during locomotion.

This species, the smallest of the pelomyxas has been found on several
occasions in large numbers in old cultures of material brought from the
marshes of Lonsdale (Pl. VIII, fig.8). Because of its dark color and rapid
rate of locomotion it at once attracts attention. Although three or four
of my cultures were very rich in this form, it must, nevertheless, be
classed as a rare species. The environment must be of a very special
kind apparently in order that it may develop in numbers. It remained
in my richest culture for about four weeks from the time it was first dis-
covered. I propose for this pelomyxa the specific name sciedti in
honor of my friend Professor R. C. Schiedt.

Under low magnification this organism appears quite black excepting
at a few small places along the sides where the color is temporarily gray-
ish owing to the accidental presence of but few of the starch grains so

THREE NEW SPECIES OF AMEBAS 93

abundant in this ameba. The posterior end also usually is light gray.
Under high powers however, the color is of a dull brownish olive green,
due to the brownish endoplasmic inclusions and the numerous starch
grains. The protoplasm is bluish green.

This species passes through very slight transformations in shape
during locomotion or at other times (fig. 10). Its general shape during
locomotion is ovoidal with the anterior end broad, while the posterior
end is narrow. Never at any time does the organism flatten itself out
to any extent on the substratum. The details of the process of locomo-
tion have not been observed carefully, but the following details may be
mentioned. Owing partly perhaps to the fluid nature of the proto-
plasm, no pseudopods are formed for the purpose of locomotion, but
broad eruptive waves of endoplasm break out somewhere near the anterior
end into which then flows a part of the animal’s endoplasm. These
waves are usually partly reflected back along the sides of the animal,
leaving a more or less clear space at the farthest point reached by the
reflected wave. Occasionally for a short period the pelomyxa may also
advance by endoplasmic streaming as is commonly observed in amebas
generally, but the larger part of its path is negotiated by eruptive waves
as described.

Another important factor in locomotion is the uroid. The animal is
not attached to the substratum anywhere except at the uroid. This is
readily observed when they are taken up with a capillary pipette. The
anterior part of the body is readily displaced by slight currents in the
water but the posterior end is not affected in this way. It seems that
the thin pseudopodium like projections of which the uroid is formed
are for the purpose of holding the organism in place and at the same
time allow it to move forward. But just how this is done could not
readily be determined owing to the fact that these uroidal projections
are very small and very transparent. It has been possible to determine
however, that the projections may be thrown out very rapidly, almost
instantaneously, so that it is possible that new projections are continually
being formed as old ones are being retracted. The alternative view is
that the uroidal projections are dragged along over the substratum
attaching themselves temporarily and locally as they pass over the
substratum. But whatever maybe the exact réle the uroid plays in
locomotion, it is evident that the organism is prevented from rolling over
by reason of its attachment to the substratum.

94 A. A. SCHAEFFER

Although there is, as has been stated, considerable uncertainty in
the direction which the waves of endoplasm take at the anterior end, the
path of the organism may nevertheless, because of the activity of its
prehensile uroid, be straight for a considerable distance. In a sense
therefore the guiding agency in locomotion is located at the posterior
end of the ameba.

The rate of locomotion in schiedti is rapid, being about 95 microns per
minute (fig. 10).

The nucleus of this species presents nothing unusual. Its shape is
spherical. The chromatin occurs in rather large masses arranged in
the shape of a hollow sphere immediately underneath the nuclear mem-
brane or at some distance from it. Usually two nuclei are present as
in the case of lentissima. The binucleate condition therefore represents
an intermediate stage between nuclear division and cell division. This
stage in these two species (schiedti and lentissima) is much longer than
in most typically uninucleate unicellulars, so that the binucleate stage
is much more common. These pelomyxas are therefore to be looked
upon not as binucleate organisms, but as typically uninucleate. The
size of the nucleus is about 7 microns. Owing to the numerous endo-
plasmic inclusions, the nuclei are difficult to see in the living condition.

The contractile vacuoles are made out only with the greatest difficulty
in normal individuals. They are seen only in the small clear areas
which are observed occasionally along the sides during locomotion. With
very attentive examination the vacuoles may then be seen. The maxi-
mum size of the vacuoles is about four microns. Nothing very definite
can be said about their number which is certainly not less than ten, but
is probably very much greater. The diastole is rather rapid. The
systole is practically instantaneous, almost as rapid as the bursting of
a bubble on the surface of water. There is a readily observed character-
istic rush of protoplasm from the immediate vicinity to the place where
the vacuole has just burst, which may be taken advantage of to locate
a bursting vacuole without actually seeing the vacuole. In this way
it is possible to determine that there are several systoles a minute in
different parts of the animal. Doubtless the fluidity of the endoplasm
is in some way connected with the small size and sudden contraction
of the vacuoles.

This species is full of what are probably glycogen grains (Stole, A.,
1900. Zeit. f. wiss. Zool. Bd. 68). Their color is a shade of olive green.

THREE NEW SPECIES OF AMEBAS 95

The shapes of the bodies are varied, mostly irregular, angular with
rounded corners and edges. The maximum size commonly met with
is about six microns. Most of them are only two or three microns long.
They are not evenly distributed throughout the body, but there seems
to be a tendency for them to collect very near the surface in what is
called the ectoplasm. In focussing along the edge one observes a serrated
outline, the teeth being represented by the protruding starch grains.
I presume that a layer of protoplasm at all times covers these bodies
when lying at the surface, though one cannot observe such a layer in
the living organism.

Besides the starch bodies there are found considerable numbers of
very small spherical bodies of a bluish green color. These are met with
in nearly all amebas, but of their nature nothing is definitely known.

The bacterial rods, the presence of which characterizes the genus
Pelomyxa, are found in considerable numbers in schiedti. The length of
these rods is about three or four microns.

The number of brownish colored inclusions which are so commonly
found in pelomyxas, is small in this species. Sometimes only two or
three masses of appreciable size are found. Very little food has been
observed in the bodies of these animals. Occasionally a diatom or a
flagellate was seen, but in the great majority of individuals no recogniza-
ble food objects were found.

When the cultures began to die out, the glycogen bodies began to
disappear gradually. In the last few surviving individuals almost no
glycogen grains could be seen. The organisms were very pale yellowish
and sluggish. Numerous large permanent vacuoles appeared. The
nuclei also changed in appearance. The chromatin receded further from
the nuclear membrane and collected itself in much larger but fewer
granules than normally. From these observations we may conclude
that the starch grains are reserve food stores ashas been shown by Stole
to be the case in P. palustris, and that the cultures died out chiefly
because of lack of food.

Zoological Laboratory, University of Tennessee.

96 A, A. SCHAEFFER

BIBLIOGRAPHY

Leidy, J., 1879. Freshwater Rhizopods of North America. pp. 324. 48 pls. Washing-
ton.
Mereschkowsky, 1879. Studien ueber Protozoen des nérdlichen Russland. Archiv.
f. Mikros. Anat., Vol. 16, pp. 153-248. 2 pls.
Parona, 1883. Essai d’une Protistologie de la Sardaigne. Arch. des Science physique
et naturelles. T. 10. Troisieme periode. pp. 225-243. 1 pl.
Penard, E. 1902. Faune Rhizopodique du Bassin du Leman. pp. 714. Numerous
figures. Geneva.
Schaeffer, A. A. Contributions to the feeding habits of ameba. Trans. Tenn. Acad.
of Science. Vol. 1. pp, 35-43. 2 figs.
1916. Concerning the species Amoeba proteus. Science, Vol. 44.

pp. 468-469.

1917. On the third layer of protoplasm in ameba. Anat. Record.
Vol. 11. p. 477.

1918. Functional inertia in the movement of ameba. Anat. Record.
Vol. 14. p. 93.

Stolc, A. 1900. Beobachtungen und Versuche iiber dié Verdauung und Bildung der
Kohlenhydrate bei einem amébenartigen Organismus. Zeit. f. wiss. Zool.
Vol. 68. pp. 25-668. 2 pls.

EXPLANATION OF PLATES

Prate VII

Fig. 2. Photograph from water color drawing of bzgemma in characteristic attitude
during locomotion. C, nuclear chromatin mass; C-S, crystals attached to spheres;
F, food mass; M, nuclear membrane; V vacuoles.

Fig. 3. Photograph from water color drawing of nucleus and excretion spheres
and crystals of bigemma. CR, chromatin granules of nucleus; C, twin crystals at-
attached to excretion spheres; M, nuclear membrane; S, spheres; Occasionally two
twin crystals are attached to each other as shown.

Fig. 4. Photomicrograph from unretouched negative of several live bigemmas
among diatoms and arcellas. The nucleus, N, is faintly shown in two of them. The
arcellas measured 58 microns in diameter.

Fig. 5. Photomicrograph from unretouched negative of several live bigemmas
in characteristic attitudes during locomotion. Note especially the blunt character
of the ends of the pseudopods. Same magnification as figure 4.

Pirate VIII

Fig. 6. Photograph from water color sketch of a P. lentissima in locomotion.
N, nucleus; CV, contractile vacuole; U, uroid.

Fig. 7. Water color sketch of a quiescent stage of lentissima. Note the numerous
and bizarre shaped pseudopods.

Fig. 8. Water color sketch of P. schiedti in locomotion. CV, contractile, vacuole;
N, nuclei; U, uroid. Note the numerous irregularly shaped starch bodies.

Fig. 10. Camera lucida sketch of a moving P. schiedti showing the slight changes
in body shape during locomotion.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL
SOCIETY VOL. XXXVII

LENKA)

SI
4
2

)

Fic. 4 Fie. 5

PLATE VII SCHAEFFER

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL
SOCIETY VOL. XXXVII

Brea Fic. 8

PLATE VIII SCHAEFFER

SPERMATOGENESIS OF THE DOG

BY

Jutian Y. MALONE

The problem of spermatogenesis has been extensively worked on in
insects and various other invertebrates. Recently several vertebrates
have been shown to possess the so-called X-chromosome which is asso-
ciated with sex-determination. Two hetero-chromosomes have been
described in most of the mammals, and they probably correspond to the
X- and Y-elements as described by Wilson (712) in the Hemiptera. Dif-
ferent members of the latter class show a variation of these elements
from forms which have only the X-element, thru those which have the
X- and Y-element of unequal size, to those in which these elements are
of equal size.

In the dog I find only one such element, the history of which I have
tried to follow in this paper. The present study has been carried on at
the suggestion of Professor M. F. Guyer, to whom I am especially in-
debted for kindly help and criticism. I wish also to acknowledge my
obligation to Dr. Elizabeth A. Smith for valuable criticism.

MATERIALS AND METHODS

Thru the courtesy of the Department of Physiology of the University
of Wisconsin, I was enabled to get the material in the living condition.
Eleven of the animals from which the tissues were taken were mongrels;
the twelfth was a thorobred bulldog. No especial differences were
noted in the material except occasional minor discrepancies in the size
of the cells.

At first samples from different regions of the living testes were placed
directly into the fixing reagents in the preliminary preparations, but the
best results were obtained when the tissues were allowed to stand for
twenty minutes before being immersed in the fixative. As experienced
by all other workers on mammals the great problem is to get the chro-
mosomes to stand out as individual elements, the tendency being for
them to mass-up before the fixative takes effect. For this reason the
following fixatives: Gilson’s, Carnoy’s, Flemming’s strong, Bouin’s,
Tower’s, Herrmann’s, Haner’s modification of Flemming’s strong, and
Allen’s modification of Bouin’s, were tried out several times in an effort

98 J. ¥Y. MALONE

to overcome this difficulty. It was found that the latter reagent gave
the best results. As urea in this solution presumably increases the
rate of penetration of the fixative, the amount of urea was varied from
one to five percent in order to determine the optimum concentration
for this tissue. A four percent solution was found to give the most
satisfactory results. The formula for this fixative is as follows: to
100 cc. of Bouin’s fluid add slowly 1.5 gm. of chromic acid and 4 gm. of
urea just before using. Heat to 37° C., add the fresh material and leave
it in the fixative for one to two hours keeping the temperature up to
37° C. For good cytoplasmic fixation put the fresh material in the
fixative at 60° C. allow to cool to 37° C. and keep at this temperature
as before. The fixative is then replaced gradually by 70 percent alcohol
until entirely removed. A convenient apparatus for this part of the
technic is described by Allen (16). The tissues are then dehydrated,
placed in xylol or chloroform, xylol-paraffin and imbedded in paraffin.
As the material fixed better after standing 20 minutes the experiment
was tried to see if allowing the tissues to autolyze from one to six hours
would improve the fixation. It was found that there was no perceptable
difference in the appearance of the nucleus altho the cytoplasm showed
evidence of being digested and the chromosomes showed a tendency to
clump up more after the first hour of autolysis.

Flemming’s strong and Haner’s modification of Fleming’s strong
gave good nuclear figures but the chromosomes did not stand out very
clearly. Carnoy’s fixative gave good spindle figures but always distorted
the cells.

The sections were cut 5 microns and 7 microns thick and were stained
with: (1) Haidenhain’s iron-haemotoxylin and counter-stained with
eosin, Bordeaux red, acid fuschin, or orange G; (2) Saffranin-lichtgrun;
(3) Saffranin-gentian violet; (4) Benda’s mitochondrial method. The
most satisfactory stain was the iron-haemotoxylin counter-stained with
acid fuschin, altho the saffranin preparations were valuable in studying
the resting stages, different phases of the growth period, and the shape
and number of the chromosomes.

Smear preparations were fixed in Allen’s modification of Bouin’s
fluid and stained with Haidenhain’s iron-haemotoxylin and Benda’s
mitochondrial stain. These preparations gave as good results in all
stages as was found in the sections and were of advantage in so far as
the cells were isolated, thus removing the danger of any confusion due
the surrounding cells or to part of the cell being cut away.

SPERMATOGENESIS OF THE DOG 99

ARRANGEMENT OF THE GERM CELLS IN THE TESTIS

The main bulk of the testis is made up of the coiled seminiferous
tubules which contain the germ cells. The seminiferous tubules are held
together by connective tissue which contains a small number of inter-
stitual cells, blood and lymph vessels. Practically all stages of develop-
ment of the germ cells may be found in a single tubule. In general the
spermatogonia and the Sertoli cells are at the periphery, then comes the
primary spermatocytes, the secondary spermatocytes, the spermatids
and the spermatozoa. ‘There is no definite seriation of the stages in any
one tubule, such as is seen for example, in the insect testes. Some
tubules are filled practically with cells in the growth stages, others with
spermatids and some with nothing but a few Sertoli cells and fibers left
by the discharged spermatozoa.

It is thus apparant that the great problem in Mammalian spermato-
genesis is to determine the seriation of the stages of development. The
criterion employed was, mainly, resemblance to the stages in forms which
had already been worked out. The size of the different cells and the
number of chromosomes were the chief guides.

SPERMATOGENESIS
S permatogonia

The spermatogonia usually lie at the periphery of the tubules, but
occasionally when rapid multiplication is taking place, they are found
in the deeper portions. The spermatogonia are hard to distinguish
from the Sertoli cells but usually they can be identified by the small
amount of their cytoplasm. The nuclei of the Sertoli cells present the
same general appearance as those of the resting spermatogonia and as
far as I can determine are the same. According to some investigators
they both have a common origin from the primordial germ cells (Heg-
ner 714),

There are present in the resting spermatogonial nuclei from one to
four large, deeply staining nucleoli which always fuse when activity
commences. This process of fusion is seen best in the saffranin prepara-
tions, which show that these bodies approach each other but before com-
ing in contact seem to be connected by thin threads of chromatin on each
side. They then completely fuse forming an oval nucleolus (figs. 1-3).
Even in the earliest periods when these bodies are well separated they
seem to be connected by thin fibers or strands of chromatin. A number

100 J. Y. MALONE

of interlacing linin threads along which small granules of chromatin are
deposited radiate from the nucleolus (fig. 4). As activity commences
in the nucleus there is a marked increase of chromatin granules about the
nucleolus as though they were threading out from it (fig. 5). At the
same time the amount of chromatin along the linin fibers increases until
the linin is practically obscured from view. Undoubtedly some of this
chromatin along the threads is from the nucleolus but the increase is
so great that some must either be synthesized or is present in the resting
nucleus in such a chemical structure that it does not react to the stains
used The cytoplasm which is made up of short interlacing fibers con-
tains in all stages a spermatosphere. This stains more intensely with
cytoplasmic stains.

As development continues the separate threads of chromatin shorten
and become thicker. These finally condense into the chromosomes of
the spermatogonial metaphase, as shown in figs. 7 and 8. It appears
as though a single thread does not become a single chromosome but
that parts of threads condense about different “centers”? which are
connected to each other by the linin fibers. The nucleolus does not
lose its identity thruout these stages but remains a large, dark body with
threads radiating from it. It does, however, approach the chromosomes
in size in the early prophase (fig. 7), but it soon becomes more darkly
stained than they (fig. 8). Cells showing the nucleolus as in fig. 7 are very
few hence it indicates that this stage is of very short duration. This so-
called nucleolus is probably the X-chromosome as it can be traced as such
throuout all the later stages by its staining reaction. The newly formed
chromosomes now take up their usual position on the spindle as twenty-
one oval-shaped bodies of which one is the X-chromosome. Information
as to the origin of spindle fibers and of the centrosomes was not obtainable
but they were very. distinct in all cases. During the formation of the
spindle, the nuclear wall disintegrates. The spermatogonial metaphase
is probably of short duration as stages where none of the chromosome
had started to divide, as in fig. 9, were very few. Counts were made
from polar views (Fig. 10), and oblique side views of the metaphase
spindles. In material obtained from a female foetus an attempt was
made to determine the somatic count in tissues taken from the pancreas,
liver, mesonephros, metanephros and kidney. A few clear spindles were
secured from liver material in which 22 chromosomes appeared (fig. 69).

In cases where the autosomes had just started to divide (figs. 11, 13,
14), it was obvious that the X-chromosome was dividing ahead of them.

SPERMATOGENESIS OF THE DOG 101

In the middle anaphase (figs. 15 and 16) the X-chromosome can be seen
still slightly ahead of the autosomes, which are now clumping together.
The early telophase (fig. 17) shows the chromosomes clumped with a
spermatosphere near each mass. The cell wall (fig. 18) constricts in
the middle of the long axis and finally divides the cell into two complete
daughter cells. The spindle fibers are still present but soon disappear
leaving the cells with a mass of chromatin, and the spermatosphere
imbedded in the cytoplasm. Thus the cells are ready for the growth
period as primary spermatocytes.

GROWTH PERIOD

Stage A-preleptotene (figs. 19 and 20). In this stage the cells from
the spermatogonial telophase have the chromosomes clumped together
in an irregular granular mass. The nuclear membrane has not yet
formed. This stage possibly represents that described by Wilson (’12)
in Oncopeltus and Lygaeus as an uncoiling of the individual chromosomes
to form separate leptotene threads, but as the chromosomes are so massed
the actual processes cannot be determined. A large black body which
is present in this mass of chromatin retains its identity throughout
the growth period and from its subsequent behavior will be called the
X-chromosome. Fig. 20 shows the leptotene threads emerging from
the chromatin mass.

Stage B-leptotene (fig. 18). The nuclear wall is now present and is
seen to enclose several thin, beaded threads of chromatin and the X-
chromosome. These leptotene threads do not form a continuous spi-
reme but appear as independent threads in both the smear and section
preparations. While the threads show no definite polarization such as
Wenrick (716) found in the Phrynotettix magnus, they form a network
which makes them very hard to count. They have not been seen to
exceed twenty in number which further indicates that they take their
origin in the spermatogonial telophase chromosomes.

Stage C-synapsis and synizesis (fig. 22). The leptotene threads of
Stage B drift toward one pole of the nucleus where they condense into
a mass. The parts of the nucleus not occupied by these threads is
clear. This stage has been studied in several forms of mammals by
von Hoff (12) who concludes that synizesis is the result of the action
of the fixative but as pointed out by Wilson (’12), Fasten (’14) and others
it occurs in the living material and thus cannot be the result of the action

102 J. Y. MALONE

of the fixative. In cases of poor fixation, however, the leptotene threads
are so contracted that their individuality cannot be made out, while
with proper fixation it is very clear that the threads do not lose their
identity during this contraction. They appear to arrange themselves
in pairs for they emerge from the mass in parallel strands.

Stage D-pachytene (fig. 23). The leptotene threads which paired
up in the previous stage now fuse side by side; that is, undergo parasynap-
sis. This is conclusively shown in this figure as in many others where
two leptotene threads can be seen fused at one end and separated at the
other. The line of fusion of these threads is not obliterated until a
much later stage. Thus the dog is another form which shows parasynap-
sis such as described by von Winniwarter (’09) Gregoire (’04), Schreiners
(04), Wilson (’12) Smith (’16), and others.

Stage E-diplotene (fig. 24). This stage is hard to distinguish from
stage D except that all the leptotene threads have fused to form the
thicker diplotene threads. In these the line of fusion of the leptotene
threads cannot be seen except in well destained preparations. The
ends of the threads appear thicker than the rest of the thread. This
stage might be called the beaded stage for each thread has the appearance
of a string of beads. They approximate the haploid number. It will
be noted that thruout these stages the X-chromosome does not lose
its identity and that the spermatosphere is present.

These diplotene threads now contract gradually into somewhat oval-
shaped chromosomes. As this contraction progresses, linin threads
connecting them appear. In the late prophase the nuclear wall breaks
down and the chromosomes take their places upon the primary sperma-
tocyte spindle. Since twenty-one chromosomes entered the primary
spermatocyte from the spermatogonial division, the leptotene threads
paired and the X-chromosome did not lose its identity, it is obvious that
the spermatocyte autosomes must be bivalent; that is, each one is made
up of two univalent chromosomes, and the X-chromosome is univalent.
This material contained no indications that the leptotene threads twisted
about each other to form chiasmas such as observed by Janssens (’09) and
Smith (16). Heterotypic tetrad figures in the late prophase stages
are not apparent although preparations stained favorably with iron-
haemotoxylin reveal a quadrapartite appearance of the bivalent chro-
mosomes as tho they were preparing for the following maturation divi-
sions.

SPERMATOGENESIS OF THE DOG 103

The actual growth period might be considered to be from stage
C to Eas there is very little change in volume up to the time of synizesis
but from there on the increase is very marked. The diplotene stage
probably lasts longer than any other as cells in this stage of development
are found in large numbers in the majority of the tubules.

REDUCTION DIVISION

When the primary spermatocytes are ready for division they reveal
ten large bivalent chromosomes, and one large X-chromosome. As the
X-chromosome lies, in the metaphase, in very close proximity to the
autosomes it is often difficult to determine its shape. But it can be
distinguished from the others as a longer, slightly curved body, (figs.
28, 29, 32, 35, 38, 39) similar to that noted by Guger (’12) and (’16).
However, a curved body from the concave or convex side would appear
as an oval body (fig. 30 and 34). Fig. 31, a polar view, presents clearly
the reduced number of chromosomes. All the chromosomes of the equa-
torial plate were not usually in the same plane.

As division starts in the autosomes the X-element can be seen to pass
slightly ahead of them to one pole. The dividing chromosomes are long
and thin giving the appearance of overlapping each other (fig. 36 and
37). This division is probably the reduction division as the autosomes
divide longitudinally and the X-chromosome passes unchanged to one
pole. In fig. 41 there could be counted at one pole, ten ordinary chromo-
somes plus the accessory while only ten autosomes passed to the other
pole. It will also be noted that the spermatosphere is still present.

INTERKINESIS

After the primary spermatocyte division no resting stage occurs.
The secondary spermatocyte metaphase is formed by the rearrangement
of the chromosomes present in the primary spermatocyte telophase.

In the late anaphase of the primary spermatocyte the centrioles with
short spindle fibers between them and the chromosome masses (fig. 44)
are apparent, indicating that the chromosomes do not reach the poles
as in the other divisions. The division of the centrioles and the forma-
tion of the new astral system cannot be followed but from the appear-
ances of such cells as figs. 46 and 47 one might infer that there is not a
new astral system formed but that the remnants of the previous spindle
are re-organized to form the secondary spermatocyte spindle. The

104 J. Y. MALONE

chromatin mass is imbedded in a more or less clear space surrounded
by cytoplasm.

As two types of cells enter this stage two kinds must result from their
division, one with ten univalent autosomes, (fig. 49), and one with ten
univalent autosomes plus the X-chromosome, (fig. 48). When the auto-
somes divide they pull apart in the center, the X-chromosome dividing
slightly ahead of them and passing to each pole, fig. 52. In the telophase
of this division the chromosomes are usually massed up but in such a
figure as 54 approximately ten chromosomes plus the accessory can be
distinguished passing to each pole whereas in others no trace of an
accessory can be found, fig. 55. Thus two kinds of spermatids which
will develop into mature sperm result from this division.

SPERMIOGENESIS

The chromosomes of the second maturation division break up into
an open reticulum composed of linin threads and chromatin granules.
The nucleus thus goes into a resting condition which apparently lasts
for some time. In approximately half of the nuclei there can be seen a
definite round body, fig. 60, which possibly corresponds with the X-
chromosome It can be seen in the nucleus after condensation of the
chromatin has occurred and the nucleus has migrated to one side of the
cell, fig. 62 and 63.

The spermatosphere, which takes the cytoplasmic stains, is present
in all of the spermatids in the cytoplasm and either imbedded in it or
closely associated with it can be seen the centrosome. In the same
region of the cytoplasm, fig. 57, is found the idiozome or remnant of the
previous spindle.

The centrosome and the idiozome are differentiated from one another
by the fact that the centrosome remains in close apposition to the sperma-
tosphere. It is a single, regular body while the idiozome is usually
lobular and irregular. As the idiozome comes in contact with the nuclear
wall an oval, clear space as described by Leplat (’10) in the cat appears
between it and the nuclear wall (fig. 58). It is apparently caused by
some repulsive force between the nuclear membrane and the idiozome
for the nuclear wall is definitely depressed. The wall of the cavity
opposite the nuclear wall is probably formed by material from the
idiozome. The nuclear wall then gradually returns to its original posi-
tion, fig. 60, the nucleus becoming longer than it is wide and the idiozome

SPERMATOGENESIS OF THE DOG 105

forming a cap over about two-thirds of its length. Later this cap be-
comes the acrosome of the mature sperm fitting closely over the anterior
end of the head. There seems to be little change in the idiozome threads
during this transformation but the dark mass at the tip disappears. In
sections threads running between the Sertoli cells and the acrosome
were usually noted but no indication of their origin could be found unless
they come from the acrosome. Fig. 68 shows a tubule from which all
of the sperm have been discharged and has nothing in it but a few Sertoli
cells with these threads running to them.

While this development of the acrosome has been going on the sper-
matosphere and the centrosome have migrated to the opposite side of
the nucleus. The spermatosphere becomes closely applied to the
nuclear wall and the centrosome divides into an anterior and a pos-
terior portion, fig. 61. At this stage the nucleus gets smaller and the
chromatin material appears as an indefinite granular mass in which a
dark body, possibly the remnant of the X-chromosome, is seen in approxi-
mately half of the cells, fig. 62. The nucleus migrates to the side of the
cell toward the acrosome apparently carrying with it a definite amount
of cytoplasm enclosed in a denser wall, fig. 62. The migration of the
nucleus is usually toward the tubule wall. At the base of this cytoplas-
mic neck which is destined to become the sheath of the middle piece of
the mature spermatozoa, is found the spermatosphere. It retains this
position until the nucleus, now the sperm head, has broken thru the
cell wall. In the “giant cells” (fig. 74) in which a number of spermatid
nuclei are present in one cell, there is a spermatosphere associated with
each nucleus.

The process of formation of the acrosome and middle piece is similar
to that found by McGregor (’99) in Amphiuma, except that he finds part
of the centrosphere or idiozome also going to form the middle piece.

After division of the centrosome the anterior one comes in contact
with the nuclear wall causing a temporary depression in the latter and
then flattens out into a disc between the walls of the cytoplasmic neck.
This disc, which forms the end knob of the sperm, has extending back
from it a thin filament which extends to the spermatosphere fig. 63.
Attached to this filament by a fine stalk is the posterior centrosome.
This condition differs from that described by Leplat (’10) in the cat and
Wodsedalek (’13) in the pig, in that the posterior centrosome in these
animals forms a ring which migrates along the axial filament and is
cast off with the cytoplasm.

106 J. Y. MALONE

As the sperm head breaks thru the cell wall the axial filament becomes
much longer. The flattened head is composed of three regions of dif-
ferent staining reaction, fig.64. The point of attachment of the acrosome
corresponds with about the posterior margin of the anterior two-thirds
of the head and the point of attachment of the cytoplasmic neck cor-
responds with the anterior margin of the posterior third of the head’
Thus it appears that the difference in the density is due to the presence
of these membranes and that the lighter middle portion is due to the
absence of these membranes. Further evidence of this is seen in fig.
65 in which the acrosome is becoming applied to the nuclear wall. Here
there are only two regions of different color. This observation may be
carried to the mature sperm, fig. 67, which shows in a side view that
there is a layer of dark staining material covering the posterior third
of the head, which also appears darker than the rest of the head when
viewed from above, fig. 66.

When the cells reach the stage shown in fig. 65 they become attached
in groups to the Sertoli or nurse cells by long filaments. At this time
the cytoplasm is cast off and the sperm continue to develop. The sur-
rounding cytoplasm is found to disappear gradually and as the sperm do
not leave the tubule until this is about complete, it is possible that they
derive some of their nourishment from this bed of cytoplasm. In the
Benda preparation there was found large and small globules of fatty
substance in this cytoplasm, in the Sertoli cells and a few in the inter-
stitual cells. These globules stain black with iron-haemotoxylin but
do not stand out as well as in the Benda preparations.

In the final changes of the sperm the acrosome becomes closely
applied to the sperm head and is no longer distinguishable. The cyto-
plasm of the middle piece contracts and slightly elongates to cover al-
most the anterior half of the sperm tail or axial filament. During this
contraction the posterior centrosome breaks thru the cytoplasmic wall
and is seen lying outside the middle piece (fig. 66). There is no evidence
of the sperm head enlarging during this process as described by Wad-
sedalek (713).

Thus the mature sperm is seen to consist of: a head formed from the
entire nucleus of the spermatid, the cytoplasm of the middle piece from
the spermatosphere and the axial filament together with the anterior
and posterior centrosomes from the centrosomes. The spermatozoa
now lose their attachment to the Sertoli cells and pass into the lumen of

SPERMATOGENESIS OF THE DOG 107

the tubule leaving behind the Sertoli cells with large bundles of threads
attached to them as shown in fig. 68.

Further evidence of the dimorphism of the sperm was obtained by
camera lucida measurements. Three hundred measurements were made
at 2,000 magnification of mature sperm, obtained from the vas deferens.
It was found that these sperm, selected at random from the preparation,
could be grouped into two classes on the basis of size. The heads of
one hundred and sixty-one at this magnification measured five to six
millimeters when projected onto paper with a camera lucida while the
balance of the three hundred measured seven to eight millimeters.
This difference in size is probably due to the presence of the X-chromo-
some.

SUMMARY

1. The five typical cells ordinarily found in spermatogenesis:
spermatogonia, primary spermatocytes, secondary spermatocytes, sper-
matids, and sperm occur in the dog in an unseriated arrangement.
These are enclosed in long, thin, winding tubules which are held together
by connective tissue and interstitutal cells.

2. Large numbers of spermatogonia and Sertoli cells are present
around the periphery of the tubules and may contain one or more large,
deeply staining bodies or nucleoli. In case of the spermatogonia these
nucleoli always fuse before activity is marked. This body is possibly
associated with or is the X-chromosome.

3. The spermatogonia show twenty-one oval shaped chromosomes,
the X-chromosome usually not being distinguishable until the early
anaphase where it divides and passes to the poles slightly ahead of the
autosomes.

4. Following the spermatogonial division the chromosomes weave
out into separate leptotene threads, while the X-chromosome remains
as a rounded or slightly oval dark-staining mass.

5. The leptotene threads undergo parasynapsis.

6. Eleven chromosomes appear in the primary spermatocyte, ten
are bivalent autosomes and one the X-chromosome. The X-chromosome
passes undivided to one pole while the autosomes divide by longitudinal
splitting. Thus there are produced two kinds of secondary sperma-
tocytes. This division is réductional.

7. There is no resting stage between the primary and secondary
spermatocyte divisions, the chromosomes retaining their identity al-
though they increase in size slightly.

108 J. Y. MALONE

8. The two kinds of secondary spermatocytes upon division give
rise to two kinds of spermatids, one with ten univalent autosomes and
the other with ten univalent autosomes plus the X-chromosome. This
dimorphism is further evidenced by the resting spermatids as approxi-
mately half of them show a large, deep staining body which is probably
the X-chromosome.

9. The chromatin of the spermatid nucleus condenses into an indif-
ferent mass, the nucleus contracts, becomes narrower and flattened. It
passes to one pole of the cell, breaks thru the cell wall and leaves most of
the cytoplasm of the cell behind. It then attaches itself to a Sertoli
cell by a thin fiber and shapes up into a mature sperm.

10. During spermiogenesis the centrosome gives rise to the end knob,
axial filament and the posterior centrosome; the sphere substance of
the secondary spermatocyte division to the acrosome; and the sperma-
tosphere to the sheath of the middle piece.

11. Measurements of mature sperm give a distinct bimodal curve,
also indicating their dimorphism.

Zoological Laboratory, University of Wisconsin.

LITERATURE CITED
ALLEN, EZRA
1916. Studies in Cell Division in the Albino Rat (Mus Norvegicus). Anat.
Rec., Vol. 10, No. 9.
FASTEN, NATHEN
1914. Spermatogenesis of the American Crayfish, Cambarus virilis and Cam-
barus dumnnics (2) with Special Reference to Synapsis and the Chroma-
toid Body. Jour. of Morph., Vol. 25, No. 4.
GREGOIRE, V.
1905. Les resultats acquis sur les cineses de maturation dans les deux regnes. La
Cellule, T. 22.
Guyer, M. F.
1912. Modifications in the Testes of Hybrids from the Guinea and the Common
Fowl. Jour. of Morph., Vol. 23, No. 1.
1916. Studies in the Chromosomes of the Common Fowl as Seen in the Testes
and in the Embryos. Biol. Bull., Vol. 31, No. 4.
Heecner, R. W.
1914. The Germ Cell
JANSSENS, F. A.
1909. La theorie de la chiasma typie. Nouvelle interpretation des cinese de la
maturation. La Cellule, T. 25.
LEPLAT, GEORGES
1910. La spermatogeneses chez la chat. Archiv. de Biol., T. 25.

SPERMATOGENESIS OF THE DOG 109

McGrecor, H.
1899. The Spermatogenesis of Amphiuma. Jour. of Morph., XV suppl.
ScHREINERS, A. and K. E.
1906. Neue Studien uber die Chromatinreifung der Geschlechtszellen. Archiv.
devbrolen ha 22-
SmiTH, E. A
1916. Spermatogenesis of the Dragon Fly Sympetrum Semicinctum (Say) with
Remarks upon Libellula basilis. Biol. Bull., Vol. 31, No. 4.
Van Hoor, LUCIEN
1912. Synapsis dans les Spermatocytes des Mammifers. La Cellule, T. 27.
von WryniwaTer, H. and Sarymont, G.
1909. Nouvelles recherches sur l’ovogeneses et l’organogeneses de l’ovarie des
Mammifers (chat). Archiv. of Biol., T. 24
WoODSEDALEK, J. E.
1913. Spermatogenesis of the Pig with Special Reference to the Accessory
Chromosomes. Biol. Bull., Vol. 25, No. 1.
WEnrRIcH, D. H.
1916. The Spermatogenesis of Phrynoteitix magnus with Special Reference to
Synapsis and the Individuality of the Chromosomes. Bull. of the
Museum of Comparative Zoology of Harvard College, Vol. 60.
WItson, E. B.
1912. Studies in Chromosomes. Jour. of Exper. Zoology, Vol. 13, No. 3.

EXPLANATION OF PLATES

All drawings were made at 1600 magnification with a camera lucida. The plates

were reduced about one-fourth.
PLATE IX

Figs. 1, 2 and 3. Resting spermatogonial nuclei.

Fig. 4. Resting spermatogonia.

Figs. 5 and 6. Spermatogonia in early stage of activity showing the increase in
chromatin along the linin fibers.

Figs. 7 and 8. Spermatogonia showing the condensation of the chromatin to
form the chromosomes of the metaphase. Fig. 8 is from a smear preparation.

Fig. 9. Side view of a spermatogonial metaphase. j

Fig. 10. Polar view of a spermatogonial metaphase showing 21 chromosomes.

Figs. 11, 12, 13 and 14. Early spermatogonial anaphase showing the chromo-
somes starting to divide.

Figs. 15 and 16. Spermatogonial anaphase showing the X-chromosome dividing
ahead of the autosomes.

Fig. 17. Early telophase of the spermatogonial division.

Fig. 18. Late telophase of the spermatogonial division.

Figs. 19 and 20. Preleptotene stage or beginning growth period. The dark
staining compact mass is the body which later is seen to be the X-chromosome.

Fig. 21. Leptotene stage showing the X-chromosome.

Fig. 22. Synapsis and synizesis showing the drifting of the leptotene threads
~ and their contraction to one side of the nucleus.

110 J. Y. MALONE

Fig. 23. Pachytene stage showing the chromsomes pairing by parasynapsis.

Figs. 24 and 25. Diplotene stage showing the beaded appearance and the unfused
threads.

Figs. 26 and 27. Late primary spermatocyte prophase showing the condensation
of the diplotene threads to form the chromosomes.

Figs. 28, 29, 30, 32, 33 and 35. Side views of primary spermatocyte metaphase,
figs. 28, 29, 32 and 35 showing the curved X-chromosome.

Fig. 31. Polar view of a primary spermatocyte metaphase showing ten autosomes
and the X-chromosome.

Figs. 34, 36, and 37. Early primary spermatocyte anaphase showing longitudinal
division of the autosomes.

Figs. 38 and 39. Parts of primary spermatocyte anaphases showing the curved
X-chromosome going to one pole.

Figs. 40 and 41. Early primary spermatocyte telophase.

Figs. 42, 43 and 44. Late primary spermatocyte telophase.

Figs. 45, 46 and 47. Secondary spermatocyte prophase.

Figs. 48 and 50. Secondary spermatocyte metaphase showing ten autosomes
plus the X-chromosome. Fig. 50 from a smear preparation.

Fig. 49. Secondary spermatocyte metaphase showing ten autosomes.

Fig. 51. Early secondary spermatocyte anaphase showing equational division.
From a smear preparation.

PLATE X

Figs. 52 and 53. Early secondary spermatocyte anaphase showing equational
division.
fe ~. Fig. 54. Secondary spermatocyte telophase showing approximately ten auto-
somes plus the X-chromosome at each pole.

Fig. 55. Same as fig. 54 except that the X-chromosome is not present.

Figs. 56 and 57. Spermatid showing the spermatosphere with the centrosome
imbedded in it and the remnants of the secondary spermatocyte spindle (idiozome).
“ 5 Figs. 58, 59, 60 and 61. Spermatids showing the formation of the acrosome from
the idiozome, the migration of the spermatosphere plus the centrosome to the other
side of the nucleus, and the division of the centrosome.

. » Figs. 62,63 and 64. Spermatids showing the migration of the nucleus to one side
of the cell, the formation of the middle piece, end knob, posterior centrosome and the
axial filament.

Fig 65. Immature sperm which has cast off its cytoplasm except that destined
to’ become the sheath of the middle piece and the fibre which seems to connect it with
the Sertoli cell.

Fig. 66. Mature sperm viewed from the broad side.

Fig. 67. Mature sperm viewed from the side.

Fig. 68. Cross section of a tubule the sperm cells of which have all matured
leaving only a few Sertoli cells and fibres.

Fig. 69. Polar view of a somatic cell of a female foetus showing 22 chromosomes.

Figs. 70 and 71. Anomalies showing heterogenic division.

Figs. 72 and 74. Giant cells showing more than one nucleus and their associated
spermatospheres.

Fig. 73. Two spermatids side by side.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL
SOCIETY VOL. XXXVII

PLATE IX MALONE

AMERICAN MICROSCOPICAL

TRANSACTIONS OF THE

AX XVII

SOCIETY VOLE:

MALONE

THIGMOTACTIC REACTIONS OF THE FRESH WATER
TURBELLARIAN, PHAGOCATA GRACILIS, LEIDY
BY
BERNOL R. WEIMER

The Turbellaria have ever been popular subjects for experimen-
tation, yet in looking over the literature, I find that very little has
been done upon the thigmotactic reactions of these animals. Thinking
that here was a problem that might yield some interesting results, I col-
lected a number of the animals in Falling Run, Morgantown, West Vir-
ginia, and these were used in the following experiments. Five specimens
were sent to Dr. H. S. Pratt of Haverford College, and were identified
by him as Phagocata gracilis.

Six rocks of varying degrees of roughness were collected and cemented
together with Portland cement in a circular glass dish 7 cm. high. The
rocks were so arranged as to form a surface as nearly flat as possible.
The outer walls of the dish were covered with heavy black paper in order
that light could fall only vertically upon the surface of the rocks.

After allowing water, which was changed frequently, to stand in the
dish for three weeks, some animals were placed in the dish. All the
animals were in good condition, but those animals which came in contact
with the cement between the rocks, secreted a slimy substance and died.
The cement was then coated with paraffin, and at the same time a piece
of glass of nearly the same area as the different rock surfaces, was waxed
in.

After carefully washing the paraffin for two days in running water,
some animals were again placed in the dish and after two days were alive
and in good condition. Forty animals were then put into the dish which
was placed beneath the skylight in a room so darkened that all the light
came from this source. The surface of the rocks in the experiments
was covered by water to an average depth of 1.5cm. The temperature
of the water varied from 21° -25° C.

In collecting the rocks used in the experiments, care was taken that
the surface should have different degrees of roughness and that each
rough surface should be as nearly uniform as possible. The rocks and
the glass plate were each numbered with India ink. These numbers
are used in the tables given, to designate the rock ard, obviously, the
surface. Rocks numbered 1, 2, 3, and 4 were of weathered sandstone
and rock number 5 of weathered lime-stone.

112 B. R. WEIMER

The surface of rock number one was composed of rounded quartz
crystals of about the same coarseness as granulated sugar. Rock number
two had a surface somewhat smoother than number one, the particles
of sand were about as coarse as table salt. Rocks numbered four,
three and six approached, in the order mentioned, rock number five, a
smooth lime stone. Thus the rock surfaces varied in coarseness or
roughness of surface from particles the size of granulated sugar to a
comparatively smooth surface in the following order: one, two, four,
three, six, and five. For a surface a grade smoother than the rocks
described, there was the glass plate and the paraffin coating the cement.
Practically the only surface difference noticed between the glass and the
paraffin was, that the paraffin had a slightly greasy feeling.

The following explanation refers to tables 1 and 2. The total number
of animals counted on a given surface during a set of observations was
found and this number, divided by the number ofobservations in the set,
gave the average observation for the set. This average observation for
each set was added and the average of this total gave the average ob-
servation for all the sets. Since only one set of observations was taken
in tables 3 and 4, the second average of the sets, was not necessary. The
area of the paraffin and the vertical glass sides of the dish covered by
water were averaged as one surface (para-glass in tables). This para-
glass surface was used as the common unit area and from this the relative
number of animals resting on a common unit surface, was computed.
Since the paraffin and the vertical glass surface was so nearly alike, both
were averaged as one. With this brief explanation, the tables given
should not be difficult to understand.

The data set forth in table 1 was procured in the following way.
The observations were taken at either ten or fifteen minute intervals
and usually after each observation the animals were stimulated, i.e.
caused to move from the place they were resting. No distinction was
made between an animal moving across and one at rest on a surface.
This probably accounts for the high averages for the various surfaces
in table 1. The dish was frequently moved around to counteract any
light reaction, tho the light apparently made no difference since it came
vertically from above. A tendency to gather in groups was also noticed.
These groups were usually found on the paraffin. The animals in these
groups were always stimulated after an observation.

THIGMOTACTIC REACTIONS OF PHAGOCATA GRACILIS 113

TABLE 1
“hog wnt | 8} 10 86) 74 tee
av. lav. jav. lav. lav. lav. jav. | av. of | No. animals | Total no. of
set1 |set2 |set3 |set4 |set5 |set6 |set7 all per unit animals
obs. surface counted
Rock
1 1.70] 1.80} 2.75} 0.70} 2.00) 4.30} 1.60} 2.13 25.53 145
2 2.40} 1.20] 1.50] 0.40} 2.55} 3.66] 0.46) 1.73 15.65 108
3 2.10} 3.40} 2.70) 1.10) 2.44) 2.50} 1.05} 2.19 18.79 249
4 0.90} 4.80} 8.25} 0.50} 2.20) 1.16) 1.90) 2.81 34.59 192
5) 2.30} 2.40} 1.66] 0.80) 1.60} 2.30) 1.90) 1.85 USA) 126
6 1.40} 1.20} 1.40] 1.30} 1.70) 3.60) 1.26} 1.66 17.66 113
Glass 7 | 1.70} 0.90} 1.00) 0.70} 1.30} 2.10) 1.20} 1.27 16.25 86
iPara-
glass |32.70|28.50/25.12|34.50/28.30)/30.30|31.86| 30.18 30.18 2053

Observations showing number of animals seen on each surface with vertical
illumination only. The time between sets varied from a day to a week. The sur-
faces in order from roughest to smoothest are as follows: one, two, four, three, six,
five and glass.

From table 1 it is seen that when all the surfaces have been reduced
to the same unit area, the three highest averages of animals resting,
are as follows, rock four, 34.59, paraffin-glass, 30.18 and rock one 25.53.
The other surfaces have much lower averages and do not vary much
among themselves. One peculiar fact to be noticed is that rock one has
the roughest surface, the glass-paraffin has the smoothest surface, and
intermediate between these two is the surface of rock four. It would
be well to remember that after each observation the animals were stimu-
lated. Since so few animals were found resting, they were averaged
with those moving.

The data in table 2 differs from that found in-table 1, in that all the
observations were taken in a photographic dark room. An electric
light was used only long enough to record the observation. The observa-
tions were taken at intervals varying from 30 minutes to an hour and the
animals moved from their resting position after each observation. As
a result of the longer interval elapsing, most of the animals were found
resting when the observation was recorded.

114 B. R. WEIMER

TABLE 2
No. of Total
obs. ; 6 6 4 20
av. av. av. av. av. of | No. of animals | Total no. of
set 1 | set 2 | set 3 | set 4 | all obs. per unit animals
surface counted
Rock 1 | 0.00 1.50 1.60 | 0.75 96 11.41 192
Dale O0n\eas:00 1.16 | 0.25 1.60 14.38 32.00
3 1009) 92.330 1 O80 2250 1.65 14.15 33.00
4 E50 et SON OlGN ee OO 1.04 12.80 21.00
Bl Os 1.66 | 6.10 1.00 2.22 18.20 45.00
GA ee SOR O50 Mrs OOU pants 2.18 23.06 44.00
Glass 7 1.50 | 2.30 5000225 1.38 17.66 28.00
Para-
glass | 32.75 | 25.60 | 27.60 | 30.75 29.17 29.17 584

Observations taken in photographic dark room showing number of animals
counted on each surface. The time between sets varied from a day to a week.

The surfaces in order from roughest to smoothest are as follows: one, two, four,
three, six, five and glass.

The averages of these observations in table 2 show, that when all
the surfaces have been reduced to the same unit area, the surfaces having
the most animals resting upon them are the paraffin-glass, 29.17, and
rock six, 23.66. Rock six has a comparatively smooth surface of weather-
ed lime-stone. The averages for the other surfaces are about the same.

After conducting the first set of experiments, the results of which are
found in tables 1 and 2, the glass was broken from around the cement
block containing the rocks. The cement necessarily exposed was covered
with a coating of bees wax. The whole block, surface downward, was
then placed upon three glass supports, 2.5 centimeters high, in an en-
ameled pan. The pan was then filled with water until it covered the
surface of the rocks. This apparatus was first put in a photographic
dark room, and observations taken each hour by lifting the rocks. The
results were so decidedly negative that only a few observations were
taken. See table 3. The animals not only did not rest upon the rock
surfaces but were found scattered in average numbers over the entire

THIGMOTACTIC REACTIONS OF PHAGOCATA GRACILIS 115

bottom and sides of the pan. Only an average number was found on
the bottom of the pan underneath the rocks.

TABLE 3
No. animals Total no.
Surface Observations Av. per unit animals
surface counted
Rock 1 Av.
2 1 0.20 1.19 1
3 2 0.40 3.43 2
4
5
6
Glass
Para.
Pan 39 | 38 | 40 | 40 | 40 | 39.90 39.90 237

One set of five observations taken in photographic dark room with the rocks
inverted, showing the number of animals counted on each surface.

The surfaces in order from roughest to smoothest are as follows: one, two, four,
three, six, five and glass.

TABLE 4

No. animal | Total no.
Surface Observations Av. per unit | animals
surface counted

ee ees ee

Rock 1
2 1 0.09 | 0.80 1
3
4
5 1 0.09 | 0.73 1
6
Glass
Para. 1 1 0.18 0.18 2
Pan 40 | 40 | 40 | 40 | 39 | 40 | 39 | 40 | 38 | 39.5 | 39.50 435

One set of nine observations taken with vertical illumination only and with
rocks inverted, showing the number of animals counted on each surface.

The surfaces in order from roughest to smoothest are as follows: one, two, four,
three, six, five and glass.

116 B. R. WEIMER

The same apparatus was then placed underneath the skylight in a
room so darkened that all the light came from this source. The same
negative results were obtained as in the photographic dark room, though
the difference was noted in that the animals in this experiment, gathered
on the pan underneath the rocks, whereas in the dark room this preference
was not shown.

According to Pearl (’02), the ventral surface of Planaria maculata
is strongly positively thigmotactic; but this does not explain the ten-
dency of the animals to rest in angles. This resting in angles he calls
goniotaxis. The physiological condition of reduced tonus helps to
determine whether or not the animal will rest on a smooth surface.

The increased resistance to movement may be one cause for stop-
ping on the uneven surface tho the most important factor is probably
light. Nevertheless, instances are known where the animal may stop
in a bright light, tho such instances are rare.

From table 1 it is seen that rock four has an average of 34.59 animals
per unit area. The surface of this rock in degree of roughness, is mid-
way between the rough granular surface of rock number one (average
25.53) and paraffin-glass (average 30.18). Table 2 shows that the
paraffin-glass surface (average 29.17) is the highest per unit area, fol-
lowed by rock six (average 23.06) whose surface is of smooth weathered
limestone. In tables 1 and 2, it will be noticed that surface number
seven is of glass. This surface is horizontal, not vertical as is that
averaged with the paraffin. If the average of this surface (16.25 in
table 1 and 17.66 in table 2) were added to the surface average of the
paraffin-glass (average 30.18 in table 1 and 29.17 in table 2), the total
average of these surfaces would be 46.43 in table 1 and 46.83 in table 2.
This is much higher than any of the rough surfaces. From this the con-
clusion may be drawn that the animals prefer a smooth surface.

Pearl (’02), in describing the method of locomotion of Planaria macu-
lata says that the ventral surface of the body constantly secretes mucus
in greater and lesser quanities. This is very sticky and under normal
conditions adheres to the surface on which the animal reposes. Thus
between the animal and the surface on which it moves there will be a
constant layer of mucus. The beating of cilia in this mucus pushes the
animal forward. Considering this explanation, then, more mucus may
be secreted by an animal when passing over a rough surface than when
passing over one that is smooth. Likewise a regular granular surface

THIGMOTACTIC REACTIONS OF PHAGOCATA GRACILIS 117

might not have quite so much effect as a very angular surface. Pearl
(02), also suggests that the reduced physiological tonus of the animal
might cause it to rest on the rough surface.

To find whether or not more mucus was extruded and secreted when
passing over a rough surface than over a smooth one, the following
experiment was tried. A piece of ordinary window glass was scratched
or grooved by a diamond glass cutter. The glass with the grooved
surface was then placed in a large petri dish and covered with water.
Some of the animals were then placed in the dish and allowed to move
over the grooved surface. After an hour the animals were removed
and the glass “developed” by immersing in Delafields’ hematoxylin,
which stained the slime blue. This gave better results than the method
used by Pearl (’02), who used a solution of carmine. This was tried
and the carmine particles by adhering to the mucus, showed the tracks
but the mucus was not stained and hence a close study of it could not
be made. The glass was then broken so that cross sections of the grooves
could be examined with the microscope. A number of sections were
thus examined but at no place could a greater slime secretion be noticed
where the slime tracks crossed a groove.

Thinking perhaps that the width and depth of the grooves in a sur-
face might cause some difference in the amount of slime secreted, the
bottom of a large petri dish was covered by a layer of paraffin to an
average depth of 5mm. In this paraffin were cut grooves varying from
0.20 to 3 mm. in width and the same in depth. The dish was then filled
with water and in it were placed some animals. The smaller grooves
did not seem to reduce the rate of locomotion of the animals. How-
ever, when an animal approached one of the wider grooves, it would
hesitate, raise up the anterior end of the body and move it to and fro,
then pass across without touching the bottom of the groove.

After the animals had been undisturbed for two hours, they were
removed and the water replaced by a solution of Delafields’ hematoxylin.
On examination of the stained slime tracks it was found that in few cases
was there a greater amount of slime secreted. The slime strands did
not descend to the bottom of the grooves but in most cases were con-
tinued on across as a sort of bridge. In some cases these strands were
broken but even then no abnormal amount of slime was found. At
the edges of the grooves, however, there were places where there seemed
to be a greater amount of slime. These were the places, probably, where
the animals hesitated.

118 B. R. WEIMER

From these results it would appear that there is the same amount of
slime on all surfaces and that no abnormal secretion is caused by a rough
surface. Thus a change in the physiological condition of the animal
as caused by the difference in mucus secretion, would not effect the
thigmotactic reaction to surfaces of varying roughness and smoothness.

Pearl (’02), suggests that perhaps the rough surface offers more
resistance to movement than does a smooth surface. This naturally
would cause a slower rate of locomotion when an animal was passing
over a rough than when passing over a smooth surface but since an equal
amount of slime is secreted on all surfaces, there should be approxi-
mately the same rate for all surfaces. Accordingly the rate of locomo-
tion was found for two surfaces, the one of rough sandstone, somewhat
coarser than rock one, and the other glass.

A piece of white paper was marked off in 1 cm. squares and placed
underneath a large petri dish filled with water. The surface of the
rock was marked off in 1 cm. squares with waterproof India ink and
around the edges were placed sides of pasteboard to keep the animals
from wandering off the surface. The rock was placed in a galvanized
pan which was filled with water to such a height that the surface of the
rock was covered to a depth of 2 cm. The two surfaces were placed
underneath the skylight in a room from which all other sources of light
were cut off.

The four animals used in these experiments were kept in separate
dishes carefully marked. When the animals were transferred from these
dishes to the surfaces, a camel’s hair brush was used in order not to
injure them. In the course of these experiments, animal number one
died, so that most of the tables show the results of the reactions of
numbers two, three and four, all of which were active, healthy, indivi-
duals. The animals were placed on the surface singly, and a number of
observations taken by means of a stop watch, as to the length of time
required to move over 1 cm. of surface. No allowance was made for an
approximate deviation of 2 mm. from a straight line in passing over 1 cm.
of surface.

The data recorded in table 5 was procured thus. A number of
observations of the rate of locomotion of each animal was taken on two
consecutive days for the glass surface and on the two following days for
the rock.

THIGMOTACTIC REACTIONS OF PHAGOCATA GRACILIS 119

TABLE 5
Av. Av.
yen Rock ist day | Rock 2nd day | rate in || Glass Ist day |Glass 2nd day | _. Bae
a mm. per) }——______________|mm. per
no. of |av. rate |no. of jav. rate san no. of jav. rate |no. of |av. rate
obs. | in mm.| obs. |in mm. : obs. |in mm.| obs. |in mm.| ‘S°
1 15 0.92 15 1.23 1.07 10 0.74 0.74
2 15 0.80 15 1.10 0.95 16 1.43 18 12 1.31
3 15 1.05 15 i LAIY/ wala 17 1.47 18 1.46 1.46
4 15 0.97 15 1.24 1.10 18 1.38 18 1.47 1.42

Observations showing the rate of locomotion. Observations taken on one
surface only each day

Table 5 shows that the average rates per second of animals number
three and four are the highest, being 1.11 mm. and 1.1 mm. on the rock
and 1.46 mm. and 1.42 mm. on glass. This shows a difference in rate
of speed per second between the glass and the rock of 0.35 mm. and 0.32
mm. Animal number two shows a difference per second of .36mm. In
these three instances the animals moved more rapidly on the glass.
However, a further examination shows that an animal in twenty-four
hours time will vary in rate of locomotion on the rough surface .31 mm.
and on the smooth .21 mm. per second. Walter (’02), who tested the
rate of speed on glass only, found that the rate varied from 1.22 mm. to °
.96 per second. So the variation in rate between the two surfaces can
scarcely be attributed wholly if at all, to the difference between the two
surfaces but to some other factor. This is further proven by a study
of tables 6 and 7.

The data in tables 6 and 7 differs from that in table 5 somewhat.
A number of observations on the rate of locomotion of an animal was
taken first on one surface and immediately on the other. Several hours
later another set of observations was taken. This lessened the chance
for so great a change physiologically as to effect the speed. The results
are seen in tables 6 and 7.

Table 6 shows at 8:00 the maximum difference of speed of all the
animals, between the two surfaces to be .16 mm. per second. At 2:00
the maximum difference is .1 mm. per second.

120 B. R. WEIMER

TABLE 6
Rock Glass
Animal time no. of obs. av. rate in mm. no. of obs. ay. rate in mm."
per sec. per sec.
2 8:30 8 NEES 7 1.21
3 until 7 0.98 y 0.92
4 11:00 9 1.17 7 1.01
2 2:00 7 0.99 9 0.99
3 until 7 1.02 9 1.12
4 4:00 7 1.01 5 1.11

Observations showing rate of locomotion. All the observations were taken on
one day.

TABLE 7
Rock Glass
Animal time { :
Nae APS ee av. rate In mm. ovo oe av. rate In mm.
per sec. per sec.
2 9:00 5 1.06 7 1.36
3 until 7 1.01 8 0.98
4 11:00 ii 1.19 8 1.24
2 1:30 5 1.03 Gf Lae
3 until Z 1.01 7 1:17)
4 3:00 7 1.19 4 1.33

Observation showing rate of locomotion. All the observations were taken
on one day.

Table 7, a day later, shows the maximum difference in rate at nine
o’clock to be .3 mm. per second and at 1:30 o’clock to be .16 mm. per
second. Further, in some cases the rate of speed is greater on the rock
than on the glass.

The difference, then, in rate of locomotion between a smooth surface
and a rough surface is so small as to be almost negligible. This further

THIGMOTACTIC REACTIONS OF PHAGOCATA GRACILIS iVA\

shows that the amount of slime extruded and secreted is not increased
for a rough surface since this would probably reduce the speed of the
animal.

According to Pearl, (02), the method of locomotion in Planaria
maculata is by means of the cilia beating in the mucus strands. Think-
ing that perhaps a study of the cilia and hypodermis might throw some
light upon the thigmotaxis and locomotion of these animals, some work
was done upon the histological structure of Phagocata gracilis.

Planarian tissue is one of the most difficult of animal tissues to study
because it is so hard to fix properly and to stain. A fixative may give
good results in one case and not in another. Five different fixing fluids
were tried, hot Gilson’s, hot and cold corrosive-acetic, twenty-five per
cent solution of nitric acid, hot corrosive sublimate and a fixative recom-
mended by Woodworth, (91), made up oi a saturated solution of cor-
rosive sublimate in fifty percent nitric acid; of these, corrosive sublimate
gave the best results. Of the various stains, borax-carmine, picro-
carmine, neutral red, picric acid and carmine (aqueous), Delafields hema-
toxylin, Ehrlich’s hematoxylin and eosin gave the best results. The
sections were cut 6.6 microns thick. In all about thirty animals were
studied.

The study was made principally of the hypodermis. On the ventral
side, figure 2, it is made up of strongly ciliated columnar epithelial cells
These contain large subcircular nuclei, nu, with evenly distributed
chromatin granules. Definite cell walls between the cells could not be
distinguished. Between the cells are spindle shaped, homogeneous
bodies, the rhabditi, rb, which (Woodworth, ’91) are developed in sub-
cutaneous flask-shaped cells which are ectodermic in origin. These were
supposed to be homologous to the nematocysts of the Coelenterata but
later were supposed to be gland secretions. Underneath the hypodermis
is a homogeneous layer, the basement membrane, bm. Under this
is found the different muscle layers and the body parenchyma, figure 1.

The dorsal hypodermis, figure 3, is made up of columnar epithelial
cells somewhat longer than those on the ventral surface. No cilia were
found on these cells except near the edges of the animal. The rhabditi,
rb, were much larger and more numerous than on the ventral hypodermis.
They were found in groups of two or three. So thick were these that
the nuclei of the cells, in many cases, were pressed out of shape and often
concealed. The nuceli, nu, of the cells, when seen, appeared to have

122 B. R. WEIMER

the same structure as those in the ventral side. Beneath the cell layer
is found the basement membrane, bm. It is somewhat wider than on
the ventral surface but has the same appearance. Beneath this mem-
brane are the muscle layers and the parenchyma, mus. par. Imbedded
in the parenchyma are numerous rhabditi mother cells, rb. me, figure 1.

According to Woodworth, (’91) these rhabditi rapidly disintergrate
when extruded into the water to form a slime which, by entangling the
prey, aids the animal in procuring food. They may also be used for
protection. Whether or not these rhabditi play any part in the locomo-
tion of the animal, I was unable to determine.

In reviewing the literature on planaria there seems to be a difference
of opinion among investigators regarding the distribution of cilia on
the surface of the animal. Pearl (02) finds none on the dorsal surface
of Planaria maculata. Metschinikow (’66) and Kennel, (79), found
cilia covering the whole surface of Rhynchodesmus and Geodesmus
but Zacharias, (’88), states that the dorsal surface of a variety of
Goedesmus is bare. Vejdowsky, (’90), maintains the same for
Microplana, the cilia in the latter cases being confined to the ven-
tral surface or sole. Woodworth, (91), found the cilia on Phagocata
gracilis (collected near Cambridge, Mass.) to cover the entire surface
of the body. I was able, tho I used the same technique as Woodworth,
to find cilia only on the ventral surface and sides of the animal. How-
ever, these animals were collected in Falling Run, Morgantown, W. Va.
This difference in localities may account for the variation in ciliation.
Mosely, ’74, explains that this absence of cilia on the dorsal surface of
Bipalium may be due to the fact that the cilia on the dorsal surface of
land planarians are weaker thru comparative lack of function and are
more easily destroyed by reagents. However this is still an undecided
question.

SUMMARY

The results in tables 1 and 2 show that there is a preference shown for
a smooth surface rather than a rough one.

Tables 3 and 4, are interesting in that they substantiate the results
found by Olmstead, (’17), that unfed Planaria maculata are positively
geotropic, that is to say, have a tendency to keep the ventral side toward
the stimulus of gravity. Likewise these results show that Phagocata
gracilis is strongly negatively phototropic.

THIGMOTACTIC REACTIONS OF PHAGOCATA GRACILIS 123

Since no mucus was found filling the grooves and cracks in the glass,
it is clear that there must be no abnormal secretion of mucus and this
is further strengthened by the results found in tables 5, 6 and 7, that there
is an almost constant rate of locomotion on both a smooth and a rough
surface. The last three tables also show that there is a variation in
rate of locomotion which may be due to the different physiological states
of the animal.

A histological study of the hypodermis discloses the fact that no
cilia could be found on the dorsal side of the animal, which may be due
to a difference in the variety of the variety of the animals, since Wood-
worth, (’91), found cilia on the dorsal hypodermis of the same species,
collected in New England.

CONCLUSIONS

1. Phagocata gracilis is positively thigmotactic to a smooth surface.

2. This is not due to varying amounts of mucus secreted.

3. Phagocata gracilis is positively geotropic and strongly negatively
phototropic.

4. The rate of locomotion is the same for both a rough and am sooth
surface.

5. No cilia are found on the dorsal surface of Phagocata gracilis
collected at Morgantown, W. Va.

University of W. Va.
Morgantown, W. Va.

REFERENCES
OtmstEaD, J. M. D.
1917. Geotropism in Planaria maculata. Jour. Animal Behavior, Vol. VII,
pp. 81-86.
PEARL, R.
1902. The Movements and Reactions of Fresh Water Planarians. Quart.
Jour. Micr. Science, Vol. XLVI, pp. 508-714.
Wa ter, H. E.
1908. The Reactions of Planarians to Light. Jour. Exp. Zool., Vol. V, pp. 35-162.
WoopswortH, W. McM.
1891. Contributions to the Morphology of Turbellaria I. on the Structure of
Phagocata gracilis, Leidy. Harvard Mus. Comp. Zool., Vol. XVI, No.
1, pp. 142.

124 B. R. WEIMER

EXPLANATION OF PLATE

PLATE XI

Fig. 1. Transverse section of the animal anterior to the mouth, X 22.
Fig. 2. Portion of the ventral hypodermis, X 2000, approximately.
Fig. 3 Portion of dorsal hypodermis, < 2000, approximately.

LETTERING

bm. basement membrane
cil. cilia

c. mus. circular muscle
hyp. hypodermis

1. cil. limit of cilia

1. mus. longitudinal muscle

rb. mc.

mus. muscle

mus. par. muscle and parenchyma
nu. nucleus

par. parenchyma

ph. pharynx

rb. rhabite

rhabdite mother-cell

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL
SOCIETY VOL. XXXVII

PLATE XI WEIMER

ADDITIONS TO OUR KNOWLEDGE OF UNIONICOLA
ACULEATA (KOENIKE)*
BY
ERNEST CARROLL FAUST

The material of Unionicola aculeata (Koenike) on which this paper
is based was secured from one of many specimens of Lampsilis luteola
Lamarck, from North Judson, Indiana. The host was determined by
Mr. Frank C. Baker, Custodian of the Natural History Museum of the
University of Illinois. The general organization of the mite conforms
to the type species described by Koenike (1890), but certain interesting
variations and certain structures yet inadequately described deserve
to be made a matter of record.

U. aculeata was originally described from Germany by Koenike, as
similar in many respects to U. crassipes (Miill.) and in others to U.
jiguralis (Koch). It differs from the former in the exact structure of the
palpus, in the number and distribution of the genital acetabula, and in
the structure of the chitinous ovipositor. Wolcott (1899) describes this
species from Michigan. The mite recorded by Soar (1899) as Ataw tav-
erneri has also proved to be U. aculeata. Piersig (1901) has made two
subspecies of U. aculeata, separating the American form, U. aculeata
sayi, from the European form, U. aculeata aculeata, on the basis of the
number of the tarsal claws, comparative lengths of the leg segments,
exact relations of the parts of the genital field, and a slight size difference.

The specimens collected by the writer conform to those described
by Wolcott in most respects. In size they belong to Piersig’s smaller
group, since the female measures about 680% long and the male
measures about 640 pu long.

Wolcott’s specimens have a terminal segment to the female palpus
which is somewhat attenuate distad, ending in four small claws. In the
writer’s material the palpus has a more distinctly thickened terminal
joint, with two finger-like claws and two thumb-like claws in the female
and with two finger-like claws and only one thumb-like claw in the male.
The penultimate joint of the palpus in Wolcott’s material has a needle
spine which is not present in the North Judson material. In the female
of this material the basal segment is in each case the thickest of the six.

*Contributions from the Zoological Laboratory ot the University of Illinois
under the direction of Henry B. Ward. No. 111.

126 E. C. FAUST

The joints are progressively narrower and longer from base to tip. In
the female there are three conspicuous spurs on the ventral side of the
basal joint, and two undivided sickle-shaped claws at the end of each
tarsus. Long needle spines are prominent on segments two to four.
Soar (1899) figures the claws of the first leg as cleft, a feature which
Wolcott (1899) describes for both U. aculeata and U. crassipes, while
those of the writer’s material are entire as in legs two to four.

Perhaps the most interesting feature of the entire body structure
of U. aculeata is the heteromorphic fourth leg of the male. On this
appendage both the joints and spines are curiously modified so that they
present a striking ornate appearance (fig. 5). The tarsus is a long atten-
uate flat:plate as in the female, with two undivided terminal claws and
two accessory spines. The main shaft of the tarsus is free from spines.
The tibia is short and thick with seven needle spines, two large heavy
spines and one small spine. The third segment has three large and three
small blunt spines, in two parallel lines at the outer edge of the joint.
The fourth and fifth joints are both shorter than joint three altho not
as short as the tibia. They are both supplied with several short bristles.
The basal joint is irregularly sculptured and bears four spines.

The genital field of the female resembles closely that described by
Wolcott, altho the cleft between the two plates is not distinguishable as
a separate structure and no posterior papillae are found in the writer’s
material. The external male genital organs (fig. 3) are very complicated,
consisting of an ornate sculpture of chitin, to which prominent muscle
bands are attached.

A thoro study of the new material and comparison with that described
by Koenike, Wolcott, and Soar, shows the wide range of variability
within the species, while at the same time it discredits Piersig’s separation
of the species into two subspecies. It seems much more desirable to
regard the species simply as highly variable rather than to maintain a
subdivision, since the lesser similarities and differences grade into each
other almost imperceptibly in various specimens.

Unionicola aculeata has been credited by some as a parasite of the
Unionidae and by others as free-swimming. Koenike (1915) has shown
that it is free-swimming during a considerable part of its life and seeks
the mussel at times of metamorphosis and propagation. This fixes our
knowledge of the periodicity of the parasitism, but leaves us in the dark
with regard to the degree of parasitism. The mites described in this

ADDITIONS TO KNOWLEDGE OF UNIONICOLA ACULEATA 127

paper were found embedded in the subdermal connective tissue of the
mantle and foot of the mussel (fig. 6). Their position was related to no
definite axis of the host. Around it was a thin tissue cyst. Inno case
were they found to have injured the host outside of the cyst. Thus,
altho an endoparasite, the evidence shows it to be only a temporary
lodger.

University of Illinois.

LITERATURE CITED
KoeENIKE, F. :

1890. Ein neuer Bivalven-Parasit. Zool. Anz., 13:138-140.

1915. Beitrag zur Kenntnis der Wassermilbe Unionicola aculeata (Koen.).

Arch. Hydrobiol. Planktonk., 10:308-319, 1 Taf.

Prersic, R.

1901. Hydrachnidae. Das Tierreich (Schulze). Lief. 13. 336 pp., 87 figs.
Soar, C. D.

1899. Atax Taverneri sp. nov.? Jour. Quekett Micr. Club, 7(2):219-221, 1 pl.
Wotcort, R. H.

1899. On the North American Species of the Genus Atax (Fabr.) Bruz. Trans.
Am. Micr. Soc., 20:193-259, 5 pl.

128 E. C. FAUST

EXPLANATION OF PLATE XII

Fig. 1. Dorsal view of female palpus, X 226; fig. 2, ventral view of first leg of
female, X 140; fig. 3, male genital organs, X 140; fig. 4, female, ventral view, X 100;
fig. 5, heteromorphic third leg of male, X 140; fig. 6, section thru outer portion of foot
of Lampsilis luteola, with encysted Unionicola aculeata, X 140.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL
SOCIETY VOL. XXXVII

PLATE XII FAUST

DEPARTMENT OF NOTES AND REVIEWS

It is the purpose, in this department, to present from time to time brief original
notes, both of methods of work and of results, by members of the Society. All mem-
bers are invited to submit such items. In addition to these there will be given a few
brief abstracts of recent work of more general interest to students and teachers. There
will be no attempt to make these abstracts exhaustive. They will illustrate progress
without attempting to define it, and will thus give to the teacher current illustrations,
and to the isolated student suggestions of suitable fields of investigation —[Editor.]

METHODS FOR STUDYING LIVING TREMATODES

Studies on the living animal are of great importance in morphological
work on the Trematoda. Such studies have been entirely neglected by
most workers on the group. I believe that an increased use of living
material in the study of trematodes will make it possible to advance the
knowledge of the group more rapidly and to avoid many errors. Mater-
ial of living larvae and adult trematodes is easily obtained for class use,
and its use adds greatly to the students’ interest. Abundant material
of sporocysts, rediae and cercariae can usually be obtained by the exami-
nation of freshwater snails and material for the study of adults from the
intestines or lungs of frogs or snakes.

In miracidia or cercariae careful studies on locomotion reveal inter-
esting specific characters, which may give hints of the type of host in
which further development is carried on. The power of extention and
contraction is so great in most trematodes, especially in larval stages,
that a true conception of size and shape can be only gained from the
living animal. The pattern of the excretory system can only be made
out from living material. In fact it is almost impossible without care-
ful studies in the living condition to define sufficiently the structure of a
cercaria to insure specific determination. The amount of detail of
structure which can be quickly obtained from the study of living trema-
todes is often surprising. In one small distome about 2 mm. in length
it was possible from one living specimen not only to work out the con-
nections of the reproductive ducts and to gain some idea, from the direc-
tion of the beat of the cilia, of the functioning of the parts, but also to
make a camera lucida drawing under the oil immersion of the connections
of the female ducts.

Agamodistomes and adults for live study should be transferred to
slides in normal saline solution and covered with thin cover glasses.

130 AMERICAN MICROSCOPICAL SOCIETY

The water should be slowly removed from the preparation with a piece
of blotting paper until the pressure of the cover glass slightly flattens
the fluke without injuring any of its structures. Such a preparation,
when carefully sealed with vaseline, will often remain a whole day in
perfect condition for study and can be examined even with an oil immer-
sion lens. Sporocysts, rediae and cercariae are usually found in large
numbers in the digestive gland of the gasteropod host. The fully
developed cercariae should be mounted for study in the water from which
the snails are obtained, and the sporocysts, rediae and immature cer-
cariae in normal saline. By slowly removing the water from beneath
the cover glass with blotting paper, cercariae so small that they are
almost invisible to the naked eye can be slowed down and flattened so
that they can be studied under the highest powers of the microscope.
When dealing with small forms it is easier to make new mounts as the
one being studied becomes too dry, than it is to try to make a prepara-
tion more permanent.

At every stage of compression different structures are brought out.
Just before a cercaria goes to pieces in the process of drying the smaller
tubules of the excretory system are distended and the movements of the
flame-cells accentuated, so that they become clearly visible. By careful
observations and the use of a number of preparations the number and
position of the flame-cells and the relation of the tubules can be gradually
traced until the whole pattern of the excretory system is understood and
recorded. By this method I have been able to work out the excretory
systems of miracidia, rediae, agamodistomes and small adult trematodes.
The pattern of one system containing one hundred and twenty flame-
cells was successfully solved. I know of no other way by which such a
complicated excretory system could be worked out. The compound
binocular microscopes recently put on the market have proven very
helpful in the type of studies described above. This instrument is easy
on the eyes and gives depth to the object observed. Several intra vitam
stains have been tried. So far no method of intra vitam staining has
been found which gives a better picture than the unstained animal.

WILLIAM WALTER Cort.
University of California.

NOTES AND REVIEWS 131

A SUBSTITUTE FOR EUPARAL

In his original paper (La Cellule, 23, 427, 1906) Professor Gilson
omitted the method of preparing this medium because of its difficulties
and referred the matter to Griibler and Holborn. Under present cir-
cumstances it will perhaps not be a breach of etiquette to submit a method
which yields a medium with similar index and properties. According to
Gilson, Euparal is a solution of Sandarac in a mixture of Eucalyptol,
Paraldehyde, and Camsal.

Paraldehyde and eucalyptol can be purchased of any chemist’s supply
house. They should be dry and neutral. If purchased from the drug-
gist, who will probably substitute oil of eucalyptus, they should be redis-
tilled, reserving the fraction, 119° to 124° for paraldehyde, and 175-177°
for eucalyptol. The druggist’s stock is in both cases about one-third
something else.

Camsal is made by taking three parts of salol and two parts of cam-
phor, and warming gently until completely liquid. Thereafter the
mixture remains liquid, but should be kept well stoppered.

Sandarac as purchased is full of dust and ants. It may be purified
as follows: 30 grams of sandarac are placed in a 200 cc. flask and 150 cc.
of absolute alcohol added. Let stand with occasional shaking until
dissolved. This mixture is rather sensitive to water vapor from the air
and should be handled accordingly. Filter the solution thru a good
filter paper into a 300 cc. flask. This is best done by resting the short
funnel in the neck of the receiving flask and covering the whole with a
bell jar under which some anhydrous calcium chloride is placed. Filtra-
tion is much more rapid than Mayer’s albumin. The receiving flask is
now fitted with a two-hole stopper. Thru one hole passes a glass tube in
which a cotton filter has been placed; to catch dust, the rubber connections
must of course be clean. ‘This filter is connected to a calcium chloride
drying tower to remove water vapor. The other hole is connected thru
a receiving flask, if the alcohol is to be recovered, and thence to an
aspirator. Air is passed while the solution of gum is warmed gently to
50-60°. Do not bubble air thru the solution, that being unnecessary and
injurious. As the solution becomes thicker the temperature may be
slowly raised to 70° and finally to 80° to remove the last of the alcohol.
When the gum is moderately brittle on cooling the operation is ended.

To the gum in the same flask, add 20 cc. Eucalyptol, 10 cc. Paralde-
hyde, 10 cc. Camsal, cork and warm gently until a homogeneous solu-

132 AMERICAN MICROSCOPICAL SOCIETY

tion is obtained. This gives a medium with an index n= 1.483 to 1.486.
The index can be raised or lowered slightly by varying the proportions
used in making the solvent. Thus, the indices of the ingredients are
about: Eucalyptol 1.456; Paraldehyde 1.39, this varies with the pre-
paration used; Camsal 1.534; Sandarac 1.525. The essence d’euparal
is, of course, the solvent mixture used above. . The green tint mentioned
by Gilson as due to a certain copper salt is probably copper abietinate
which can be had of Merck or can be made of sufficient purity by any
student in the organic chemistry laboratory,

In my experience there is less difficulty in preparing this medium

than with some of the staining mixtures. It takes time but also little
attention. Slides mounted several months ago are in excellent condi-
tion and as near as one can judge the medium acts like Euparal. Sec-
tions can be mounted from 80% alcohol, either with or without passing
thru the essence.
E. S. SHEPHERD.
Geophysical Laboratory.
Washington, D.C.

CHROMOSOMES OF RANATRA SP?

During the summer of 1914 while working on the male germ cells of
another type, I prepared and sectioned some testes from a species of
Ranatra collected about Madison, Wis. The large number of chromo-
somes together with what seemed to be a very puzzling polymorphism
of spermatocytes induced me to defer a further investigation till a later
time. Recently the work upon this form has been resumed and has
progressed to a point where a preliminary and tentative statement may
be made.

The testes in the later nymph stages are especially valuable for sec-
tioning as they present in many cases the whole history of the germ cells
from the last spermatogonial divisions to mature spermatozoa. Sex
organs from adults collected in the spring, and up to mid-summer are
also generally favorable, but specimens taken in late summer and fall
show very few division stages.

My first material was composed of several testes from animals col-
lected in mid-summer and at that time believed to belong to but one
species. All of these were prepared together for study. Observations

NOTES AND REVIEWS 133

upon this mixed material show that there are at least two types of testes
as regards the chromosomes.

The first type has spermatogonia with 40 chromosomes of various
sizes; primary spermatocytes with 21, all of which divide equally; second-
ary spermatocytes with 21, two of which do not divide but pass directly
into different spermatids each of which then possess 20 chromosomes.
The two chromosomes which do not divide in the second division act
as a typical XY pair and always appear near the center of the chromosome
group in this division but their behavior is not sufficiently different to
enable them to be identified before this stage.

The second type has spermatogonia provided apparently with 8 or
10 more chromosomes than the first type. The primary and secondary
spermatocytes seem to have as a distinguishing mark a group of very
small chromosomes near the center of the larger group. Neither the
number nor the behavior of the chromosomes in the spermatids has been
determined although some interesting conditions are suggested by the
rather meager observations made to date.

Another interesting though not necessarily important fact is that
among the individuals collected in July few possessed testes of the
second type while a large percentage of those collected in September did
possess cells of that type.

In addition to the interest attached to spermatogenesis these forms
seem to offer an opportunity to determine possible correlation between
the chromosome differences and somatic variations as soon as the indivi-
dual origin of the two kinds of germ cells can be determined.

A. M. CHICKERING.
Beloit College.

NOTES ON COLLECTING AND MOUNTING ROTIFERS

C. F. Rousselet, the veteran English naturalist (J. Q. M. C., Nov.
1917) sums up methods which he has worked out for collecting,
handling, preserving and mounting rotifers.

For a collecting stick he recommends a walking stick with a telescopic
joint, with a ring net 6x5)4 inches and 6 inches long, made of bolting silk
No. 15 or 16. Silk lasts longer than mull and does not clog or shrink as
it does.

134 AMERICAN MICROSCOPICAL SOCIETY

The bottled materials collected are placed, on reaching home,
in aquaria with 7x7 inch parallel sides one and one-fourth inch in
depth from back to front. After a few hours the debris will have
settled, and if a strong light be placed at one face of the aquarium the
free-swimming rotifers will collect on the side toward the light, and can
be discovered with a lens and be picked up with a pipette. In solid
watch glasses these general collections can be examined quickly for new
species with the low power of the binocular and unfamiliar forms trans-
ferred to the live-box or the micro-glass trough for special study.

Narcotising the mass of rotifers in the watch glass is readily effected
by 1% cocaine. They may be killed and fixed by a drop of 4 to %%
osmic acid. They should be exposed to the osmic acid for a minute only
and then removed to formalin of 214% strength, changing it several
times until well washed.

The sorting out of different species is done under the binocular by
means of a bristle mounted in a suitable handle. They are then picked
up with a fine pipette and placed in an appropriate micro-cell, and finally
mounted in 214% formalin.

The ringing of the micro-cells may be done as follows: first a thin
ring of picture copal varnish; then several coats of Heath’s cement
(gold size-shellac-India rubber); finally finishing with three more coats
of gold size.

METHODS OF PRESERVING CERTAIN MARINE BIOLOGICAL SPECIMENS

F, Martin Duncan (J. R. M. S., Dec. 1917) brings together methods
which he has found most practical and successful in preserving marine
plant and animal life and in preparing it for microscopic examination.
Many of these methods are standard; but summarizing some of them may
be of value.

Anaesthetising

Place the smaller and specially sensitive medusze in just sufficient
sea-water for free expansion and swimming, and add two drops of 1%
solution of hydrochloride of cocaine, gently stirring with a glass rod.
Repeat at five minute intervals until the tentacles do not contract when
gently touched. Add 10-20 cc. of 4% formaldehyde solution, stirring
for several minutes. Store in 10% formaldehyde. Do not allow speci-
mens to remain in cocaine longer than absolutely necessary before adding
the formaldehyde, as the former softens the jelly of the medusz.

NOTES AND REVIEWS 135

~The author regards cocaine as, on the whole, the best anaesthetic for
the most of the smaller forms of marine life. Solutions of coacine must
be made anew since they do not keep well—becoming filled with fungoid
growths.

Hydroid zoophytes, simple and compound ascidians, holothurians,
anemones, and the like may be stupefied effectively with menthol.
This is slow in action and does not simulate to contraction. The animals
are submerged in clean sea-water and methol crystals are strewn over
the surface. Their solution is slow, and in twelve or twenty-four hours
depending on the size and sensitiveness of the animals and the amount
of water, the specimens will be narcotised in an extended position, and
may then be killed and fixed in any suitable fluid.

Fixing

The author prizes Bouin’s fluid (Picric acid, saturated aqueous solu-
tion, 75 parts; formalin 25 parts; glacial acetic acid, 5 parts) as the best
fixative for histological purposes. It has great power of penetration,
kills quickly, and fixes well. It allows after treatment of the most varied
sort. Next in desirability he considers saturated solution of corrosive
sublimate.

Weak osmic acid (a few drops of a 44% solution added to the water
in which the organisms are) is suggested for marine Protozoa. Radio-
laria are effectively killed and fixed in corrosive sublimate. Spherozoa
give good results in equal parts of sea water and 70% alcohol with a
trace of tincture of iodine added.

For Echinoderm larve an exposure of four minutes to a cold satura-
ted solution of corrosive sublimate is recommended. For whole mounts
dilute cochineal stain—as Mayer’s alcoholic cochineal formula.

Small sponges are placed, on collection, in 1% solution of osmic acid
and left there for five minutes, then transferred to strong alcohol and
changed twice. Stain sections in Mayer’s alcoholic cochineal.

Compound ascidians with contractile zooids may be handled to
advantage by placing in clean sea water, narcotizing with methol,
and then plunging for three to ten minutes in glacial acetic acid. Wash
in 50% alcohol and pass thru successive grades to a preserving strength.
Use no metal in the operation.

For small crustacea, both larve and adults, first treatment with 5%
formaldehyde in sea water is recommended. Transfer to 70% alcohol.

136 AMERICAN MICROSCOPICAL SOCIETY

For demonstration mounts it is necessary to guard against overstaining.
Weak alcoholic picro-carmine cleared in turpineol is advocated as a
means of staining.

THE SILVERMAN ILLUMINATOR FOR MICROSCOPES

This illuminator, invented by Professor Alexander Silverman of the
School of Chemistry, University of Pittsburg, is a small, circular tube
lamp which can be fitted quickly to any objective. It moves up and
down with the barrel and furnishes a diffused and uniform illumination
at the exact place where it isneeded. It is suitable both for low and high
power work, and may be used both for direct examination and for photo-
graphy of opaque objects.

Much structural detail is revealed by this device which the older
forms of illumination do not give. It isa low voltage tungsten lamp, and
may be supplied either in colorless glass or in daylight (blue) glass.
Its life is about 100 hours. There is no image of the source of illumina-
tion nor does the light strike the front of the lens except as reflected from
the object.

The intensity of the light reaching the eye is lower than in other
types of illumination, and yet because it is directed upon the spot ob-
served the observer sees more. There is no glare, no waste light, no
unduly contracted pupil, no unnecessary eye strain.

The lamps are manufactured by Ludwig Hommel & Co., Pitts-
burg, Pa.

TRANSACTIONS

OF THE

American
Microscopical Society

ORGANIZED 1878 INCORPORATED 1891

PUBLISHED QUARTERLY

BY THE SOCIETY

EDITED BY THE SECRETARY
T. W. GALLOWAY

BELOIT, WISCONSIN

VOLUME XXXVII
NUMBER THREE

Entered as Second-class Matter August 13, 1918, at the Post-office at Menasha,
Wisconsin, under act of March 3, 1879.

Che Collegiate Press
GrorcE Banta PuBLISHING CoMPANY
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1918

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at Winona Lake, Ind., 1903
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at Sandusky, Ohio, 1905
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at Minneapolis, Minn., 1910
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at Washington, D. C., 1911
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at Cleveland, Ohio, 1912
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at Philadelphia, Pa., 1914
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at Columbus, Ohio, 1915
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at Pittsburg, Pa., 1917

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TABLE OF CONTENTS
FOR VOLUME XXXVII, Number 3, 1918
Observations on Reproduction in Certain Parthenogenetic and Bisexual Nema-

todes Reared in Artificial Media, by Paul S. Welch and L. P. Wehrle........ 141
A New and Remarkable Diatom-eating Flagellate, Jenningsia Diatomophaga,

Nov. Gen., Nov. Spec., with Plate XIII; by Asa A. Schaeffer...................0000 177
Studies in American Stephanophialinae, with Plates XIV, XV; by Ernest Car-

POU SAGE: 4 tae iiadhy casio ctuantchaenehoecacioeena eect escke chav Alp asteree aera or 183

_ Notes and Reviews: Aquatic Microscopy for Beginners (Stokes)............:c:c:csessesese 199

Necrolory Dr Albert McGCalla 2 2 oe re Oe eee \ cians 201

TRANSACTIONS

OF
American Microscopical Society

(Published in Quarterly Instalments)

Vol. XXXVIT JULY, 1918 No. 3

OBSERVATIONS ON REPRODUCTION IN CERTAIN PAR-
THENOGENETIC AND BISEXUAL NEMATODES REARED
IN ARTIFICIAL MEDIA*

By Paut S. Wetca# and L. P. WEHRLE

CONTENTS PAGE

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*Contribution from the Entomological Laboratory, Kansas State Agricultural
College, No. 33.

142 WELCH AND WEHRLE

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INTRODUCTION

Nematodes present material for a wide variety of investigations.
Not only is the group very incompletely known, especially the free-living
species, but much also remains to be learned about their life processes.
One of the fertile, outstanding possibilities for investigation relates
to the remarkable features of reproduction presented by these animals.
Maupas (’00) and others have pointed out the striking diversity in the
methods of reproduction and the possibilities of this group in extending
the knowledge of sex determination, origin of parthenogenesis, origin
of bisexuality, and the nature of hermaphroditism. The development
of methods whereby these animals can be maintained in pure cultures
for indefinite periods adds to their value as material for critical study.
In this paper, the writers present results of observations on cultures
maintained continuously for more than three years. The data herein
presented are general in character and the results of study of certain
special problems will appear at a later date.

The writers wish to express their indebtedness to Dr. N. A. Cobb for
the identification of the specimens used in this work and to Mr. A. L.
Ford who, during the absence of the writers, maintained the cultures
through the summer months of 1917.

METHODS OF CULTURE

In connection with the work on which this paper is based, the writers
had occasion to use a number of methods for rearing nematodes in arti-
ficial media. Previous investigators devised means whereby certain
free-living and parasitic nematodes could be maintained for a number
of generations under laboratory conditions and some of their results
were used to advantage in the present work. In some cases, previously
known methods have been modified, while in others, new procedures
were developed. It is obvious that, in order to study these animals

REPRODUCTION IN CERTAIN NEMATODES 143

successfully, they must be reared under conditions other than those of
the natural environment and the fact that they can be cultured in arti-
ficial media for indefinite periods of time makes it possible not only to
observe continuously the details of activities and of life history but also
to carry on a large variety of experiments. The writers maintained
continuous cultures of Diplogaster erivora and Cephalobus dubius for
over three years. Cultures of the latter are still under observation
and are all descendants of one original stock. Since the published
methods of rearing the smaller nematodes appear in scattered sources,
the more important ones will be given brief notice here. In addition,
the methods employed by the writers will be discussed in detail.

METHODS OF OTHER INVESTIGATORS

Conte (00a, p. 374) cultured Rhabditis monohystera by employing
as nutritive media “la colle de pate trés epaisse les solutions de peptone
et les tranches de pomme de terre. Ces cultures etaient faites dans
des assiettes couvertes et, d’autre part sur porte-objets en isolant un
femelle fécondée dont j’etudiais ensuite la descendance.” He was
also able (’00b, p. 376) to rear Diplogaster longicauda “dans la colle de
pate.”

Metcalf (03, pp. 93-98) reared a nematode of uncertain identity
(Rhabditis brevispina ?), found in diseased corms, young stalks of Crocus
and cuttings of Petunia, Coleus, and Geranium on plates of asparagus
agar. Sterile agar was unsuitable, but a one per cent asparagus juice
agar, inoculated with a Fusarium and the bacteria which occurred with
the original stock of the nematodes and allowed to decay for about
two weeks, was satisfactory after it had been heated, filtered, and steri-
lized. In this medium, sterilized eggs developed rapidly and normally.
A certain fluidity of the medium was found to be desirable.

Potts (’10, pp. 443-446) readily cultured certain free-living nematodes
(Diplogaster maupasi, Rhabditis gurneyi, Rhabditis elegans, Rhabditis
duthiersi, Rhabditis sechellensis) in drops of nutrient media in watch-
glasses closed with a vaselined glass cover. Solutions of “brown” and
“white” peptone, used almost exclusively as the media, were allowed
to putrify until a cloudy growth of bacteria had formed throughout them.
In the presence of large numbers of bacteria, the nematodes thrived

. but, in sterile solutions, growth was suspended and eggs were deposited

144 WELCH AND WEHRLE

only at long intervals. Potts did not record the strength of the peptone
solutions—a matter of importance since, as will be shown later, an excess
of peptone has a deleterious effect on the worms. Two or three successive
generations were reared in a saturated solution of gelatin in water, and
mature individuals were secured from eggs in “solutions of amides like
tyrosin and leucin” but restriction of both growth and egg production
indicated that these media were inferior to peptone. Beef infusion was
also used to a very limited extent.

Oliver (’12) cultured an unknown nematode, found in the exudate
about the genitalia of dead guinea pigs, in a medium made by inoculating
a little of this exudate onto moist earth and slants of Musgrave’s amoeba
agar, kept at a temperature of about 75° F. Successful subcultures
were also made by using plain agar and ascites agar, plain agar and
amoeba agar giving the best results.

Johnson (’13, pp. 612-618) was able to culture Rhabditis pellio, a
parasite of the nephridia of the common earthworm (Lumbricus terres-
tris), by methods somewhat similar to those used by Potts, Conte, et al.
Filtered tap water or salt solution in watch-glasses to which were added
certain food materials constituted the basis of the cultures. The watch-
glasses were either covered individually by means of a vaselined glass
cover, or else several, without covers, were placed in a humid chamber
which gave a larger air space but minimized evaporation. The culture
medium was replenished from time to time. Experiments with Witte’s
peptone showed that a nearly saturated solution quickly proved fatal
to the nematodes and the same result accompanied weaker solutions
until a 0.15 per cent strength was reached. Although very dilute, this
solution proved satisfactory for one series of cultures, but afterwards it
was almost uniformly unsuccessful. A one per cent. hay infusion was
successful for only one series. Meat extract, decaying meat, a solution
of urea, and a hay infusion sterilized and inoculated with soil bacteria
were all virtually useless. Decaying earthworm had an advantage over
peptone in nutritive value, but presented the disadvantages of requiring
special treatment to prevent introduction of nematodes already present
in the worm; of becoming increasingly opaque as decay advanced, thus
rendering examination difficult; and of occasionally becoming foul
smelling and densely clouded, causing the death of all of the nematodes.
The best medium was prepared by removing the alimentary tract from
freshly killed Lumbricus terrestris and placing the body in a test tube

REPRODUCTION IN CERTAIN NEMATODES 145

which was then plugged with cotton-wool, heated in a steamer and kept
at a boiling-point for about two hours, decanted, filtered, and inoculated
with the bacteria which were associated with the worm in the soil. These
bacteria were secured by allowing a fresh Lumbricus terrestris to decay in
a small amount of water to which a little earth had been added. This
medium also possessed the disadvantage of occasional development of
bad odor and opaque scum, leading to the death of the entire culture.

Byars (714) made use of a synthetic medium, Pfeffer’s nutrient agar,
in connection with some studies of the plant nematode Heterodera radici-
cola and found it suitable for the preliminary development of the worms
up to their penetration into the tissues of experimental seedlings grown
in the same medium. Most of the individuals which remained outside
of the root of the seedling died within a few days, although a few
remained active for more thana month. The latter, however, did not
undergo their normal development into adults. It will be shown later
that this medium is much more successful for certain other species.

Merrill and Ford (’16, pp. 121, 126) found that Diplogaster labiata
flourished in water cultures to which had been added portions of the
macerated bodies of the beetle Saperda tridentata—the host of this nema-
tode. They state that “Different substances were tried with varying
success, but macerated beetles placed in water seemed to be the most
satisfactory”’ but these “different substances” were not specified. They
also reared Diplogaster erivora in similar preparations.

METHODS OF THE WRITERS
Equipment

The writers had occasion to employ a variety of culture methods
demanding a corresponding variety of equipment. Most of the cultures
were maintained in cells constructed as follows: Cylindrical glass mount-
ing-cells with ground edges were sealed to ordinary glass microscope
slides by means of pure vaseline and covered with a circular cover-glass,
the diameter of which was slightly greater than that of the glass mount-
ing-cell. The cover-glass was also sealed on with vaseline. For stock
cultures, the most suitable glass mounting-cell was found to be one having
a diameter of 20 mm. and a height of 10-15 mm., preferably 15 mm.
These tall cells made it possible to maintain a greater volume of the
liquid culture medium, thus providing for a larger stock and, more im-

146 WELCH AND WEHRLE

portant still, minimizing the danger of cultures drying out since it was:
discovered that they could not be sealed tightly but demanded at least
a small opening at the top. They offered the disadvantage of being
too high for examination with anything but very low powers of the com-
pound microscope. It was possible, however, to use the binocular
microscope, and since there was little occasion to use the stock cultures
for purposes other than maintenance, this disadvantage was of little
consequence.

Cultures maintained for experimental or detailed observational work
were kept in glass mounting-cells of another size, viz., diameter, 20 mm..,.
height, 6 mm. Such a cell surrounded a space large enough to enclose
a drop of the culture medium without involving the danger of the latter
coming into contact with the vaselined edges of the cell, a precaution
which had to be observed rigidly. That height also permitted examina-
tion with the ordinary low powers of the compound microscope without
removing the cell from the supporting slide.

Many of the glass mounting-cells used in this work have walls 1 mm.
thick. Cells having walls of 2 mm. thickness were found more advan-
tageous, especially for stock cultures, owing to the greater vaselined
area in contact with the slide, thus minimizing the danger of the watery
medium leaking out and permitting the culture to dry up. The thinner
cells were in more constant use owing to the difficulty of obtaining the
thicker ones, and, if care was taken to thoroughly vaseline the ground
edges and press them down onto the slide, they were quite satisfactory.

In special cases when greater space or capacity was desired, small
stender dishes were used. A limited use was made of a group of glass
mounting-cells sealed to the bottom of a Petri dish. For rapid observa-
tion of isolated individuals, Petri dishes alone were used for one series,
large drops of the nutrient medium retaining their identity when placed
on the dry, clean glass but not in contact with each other.

At times, culture slides of two kinds were used in place of the glass:
mounting-cells mentioned above. One form is the heavy plate glass,
76 x 26 mm. slide, 5 mm. thick and with a central, circular depression
4 mm. deep, and covered by sealing on a circular cover-glass with vase-
line. This form of receptacle was satisfactory in many respects but
was not usable for individual cultures because of its depth. Further-
more, it is more expensive than the glass mounting-cell. The other
culture slide is the ordinary 76 x 26 mm. form, having a central, shallow

REPRODUCTION IN CERTAIN NEMATODES 147

concavity with maximum depth of about 1 mm., closed by a vaselined
circular cover-glass. This type is useful for individual cultures.

Needles for transferring eggs, larve, and adults from one culture
to another were in constant use. These instruments were made by
setting into a small handle a fine needle, the point of which had been
bent into a small recurved hook. It was found convenient to have
several of these needles whose points were bent at different degrees.
Ordinary insect pins of sizes 0 and 00 were used to some extent but the
most useful form was the Japanned steel “Minuten Nadeln” commonly
used for pinning minute insects. These pins made especially useful
needles because of their very small diameter and extremely fine points,
thus facilitating their manipulation under the high power of the bino-
cular microscope and their use in transferring the minute nematodes.

Transferring brushes, which for part of the work were used instead
of needles, were made from small camel’s hair brushes by carefully cut-
ting out the brush until a very small central tuft of 3-5 hairs remained.
Such a brush was often used for transferring eggs.

Pipettes of the ordinary form were in common use for handling the
various fluids employed in the work. In order to avoid contamination
of the cultures, it was necessary to keep these pipettes properly labeled
and thoroughly clean. A special form of pipette for removing specimens
or for removing deteriorating culture fluids was made by drawing out
in a flame the open end of an ordinary straight pipette until the opening
was only about 0.3 mm. in diameter.

Media

In culturing nematodes, it is necessary to use some substance which
will serve directly or indirectly as food for the animals. The writers
have tried out a number of substances some of which have been distinctly
successful. Since the same substances were not in every case employed
for both of the species reared, the culture methods of each will be dis-
cussed separately.

Practically all of the culture media were more favorable under con-
ditions of dilution, in fact, some of the most successful ones demanded
extensive dilution, otherwise they were inimical to the worms. Dis-
tilled water was uniformly used, thus eliminating the danger of contami-
nating the cultures from that source, as would be the case with tap-water.
Preliminary experiments with both Cephalobus dubius and Diplogaster

148 WELCH AND WEHRLE

erivora showed that all stages of the life history lived for.some time,
several days in many cases, in distilled water alone, and when a small
amount of nutritive substance was added, the medium became suitable
for continuous culture. Apparently, the usual toxicity of ordinary
distilled water had no deleterious effect on the worms.

Diplogaster erivora.—Since the original stock of. this species was
found in the eggs of grasshoppers, the yolk of these eggs was at first
used as a medium and, as might be expected, gave good results. Eggs
from a number of species of grasshoppers appeared to be of equal value.
Owing to the occasional difficulty or inconvenience in getting grasshopper
eggs, the eggs of certain Coccinelide, of the Colorado potato beetle, of
certain unidentified insects, and the ovaries of grasshoppers were success-
fully substituted. Furthermore, the softer tissues of young grasshopper
nymphs, army worm pupz, variegated cutworm pupe, Hessian-fly
pupz, and pupz of a number of other insects were found to constitute
good media when prepared in the proper way. It is very probable that
a wide variety of insect eggs and tissues would serve this purpose equally
well.

The procedure in the use of the above mentioned substances depended
upon the kind of culture desired. Most of the stock cultures, as well as
the majority of the life history cultures, were maintained in the glass
mounting-cells already described. When grasshopper eggs were used,
they were first carefully cleaned externally and stripped of their shells,
since fragments of the latter when present tend to render the examination
of the culture more difficult. The contents were placed in a small
amount of distilled water and thoroughly triturated in order (1) to dis-
tribute more uniformly the yolk throughout the culture and render it more
readily available to the nematode, and (2) to facilitate the examination
of the material for evidence of previous nematode infestation, thus aiding
in the maintenance of pure cultures. This precaution is necessary since
small nematodes often occur on the surface and apparently within some
of these insect eggs and pupx. The writers found them in connection
with Hessian-fly puparia, both on the surface and apparently in the
pup, although in the latter case there is the possibility that they might
have been included by manipulation. However, Marchal has reported
Osborn, ’98, p. 41) nematodes in the puparia of this insect. Insect
tissues mentioned in a foregoing paragraph were sometimes used in a

REPRODUCTION IN CERTAIN NEMATODES 149

similar way, removing, of course, all fragments of the integument. The
proper amount of the diluted nutriment was placed in the glass mounting-
cell which already contained the appropriatc amount of distilled water.

The method of adding the food materials thus prepared was deter-
mined by the purpose of the culture. For stock cultures, two or three
drops from a pipette were added every 3-5 days to the 1-1.2 c.c. of dis-
tilled water in the mounting-cell. A larger amount of food could be
added without serious results to the organisms but it was not needed for
nutritional purposes, and it had the disadvantage, at least in certain
cases, of tending to increase the opacity of the culture.

When there was any occasion for using large quantities of the material
for a medium, good results were secured by putting sterilized soil in a
covered stender dish, moistening it liberally with distilled water, and
transferring to it a pupa or other food object which was then perforated
and inoculated with the nematodes from a stock culture. These cul-
tures lasted for considerable periods of time, developing nematodes in
quantities.

While the above-described methods were satisfactory, it was incon-
venient in the maintenance of a long series covering different seasons of
the year to keep a constant supply of the food materials at hand and to
have a continuous stock of any one kind for experimental purposes. For
that reason, a search was made for some food which is approximately
constant in composition and easily available at any time. Among
other things, the yolk of hen’s egg was tried and found successful. Ex-
periments with different amounts of this substance showed that, in a cul-
ture containing 1-1.3 c.c. of distilled water, the nematodes thrived when
the quantity represented by three or four dippings of the points of ordin-
ary forceps into the yolk was added. Almost any larger amount may
be used but increased opacity and the occasional undue putrefaction
make it undesirable. Amounts of food represented by one or two dippings
of the forceps were, in general, found to be distinctly inferior in results
and evidently represent too poor a culture. A single hen’s egg supplied
food material for a number of days. A small opening, approximately
1 cm. in diameter, was made through the shell and the adjacent albumen
allowed to escape. Sterile forceps were used to lift out the desired
quantities of the yolk and then the opening in the shell was sealed over
with a gummed label or piece of gummed cloth and the egg kept in a cool

150 WELCH AND WEHRLE

place. When needed again, the gummed covering was removed, the
desired quantity of yolk secured, and the opening again sealed. The
amount of yolk adhering to the points of forceps is variable but the
method was found to be both rapid and practical in the operation of
ordinary cultures.

Liebig’s extract of beef, diluted to different degrees with distilled
water, was tried as a nutrient medium and found to be usable so far as
maintaining the animals was concerned but its partial opacity impaired
its usefulness.

Since Byars (’14, p. 323) found Pfeffer’s synthetic agar useful in
culturing certain nematodes parasitic on plants, it occurred to the writers
to experiment with it as a possible medium for rearing Diplogaster erivora.
The medium used was as follows: calcium nitrate, 4 grams; potassium
nitrate, 1 gram; magnesium sulphate, 1 gram; potassium dihydrogen
phosphate, 1 gram; potassium chloride, 0.5 gram; ferric chloride, trace;
distilled water, 6 liters; and agar, 12 grams. The formula used by Byars
calls for powdered agar but the writers used the shredded agar cut up
into small bits. This fluid was used directly from the stock solution
without change and, while it appears from all the trials that it is some-
what inferior to certain other media used, nematodes were maintained in
this solution for long periods of time. According to the experience of the
writers, cultures in this medium, when properly cared for, are fairly
satisfactory, but since there was some evidence of inferiority, parallel
series of tests were carried on for 20 days, comparing the relative merits
of Pfeffer’s solution and hen’s egg as media. Using the rate of develop-
ment and reproduction as indices of the value of the media, four of the
five series showed evidence of the superiority of the hen’s egg, one of the
five showing a slight advantage for the Pfeffer’s solution.

Maceration cultures used by the writers have already been described
and it seems very probable that, barring the danger of contamination,
they might be effective and that a number of different animals could
be used as sources of food supply. Mr. A. L. Ford reared Diplogaster
erivora for one year on the macerating bodies of adult Saperda tridentata
and Hydrophilus triangularis. He also found it possible to use macerated
beef but the cultures proved somewhat unsatisfactory because of the
offensive odor. This nematode was found by Merrill and Ford (’16)
parasitizing termites and Mr. Ford has informed the senior writer that
he tried the macerating bodies of these insects as a medium but found it

REPRODUCTION IN CERTAIN NEMATODES 151

unusable since a mould almost invariably appeared in the cultures, filling
them with threads and obscuring the entire preparation. The writers
also tried macerated termites with similar lack of success from the same
cause.

Cephalobus dubius——Maupas (00, p. 611, 613) found some difficulty
in culturing the parthenogenetic nematodes, including this species, stat-
ing that “elles se prétent mal 4 des cultures en grand.”” However, the
writers have been able to culture Cephalobus dubius in countless numbers
at any time. Since studies of this species were not begun until cultures
of Diplogaster erivora had been in progress for about two years, the
methods which had proved most satisfactory were tried for Cephalobus
dubius and were likewise found to be successful. Several series of cul-
tures were maintained in Pfeffer’s solution but many of the life history
studies were made using the hen’s egg preparation. Both media were
used as described in the preceding section, and both proved suitable for
prolonged cultures, indications pointing to the hen’s egg being slightly
preferable. Grasshopper eggs, Colorado potato beetle eggs, and pupz
of Hessian-fly also proved suitable but were not always readily available.

As has been mentioned, other workers made some use of peptone
as a nutrient medium for the culture of nematodes. After a number
of trials with different strengths of peptone, a solution was found which
has proved thoroughly satisfactory and, at the present writing, cultures
of Cephalobus dubius have been running in this medium for months, with-
out any indication of weakness. The strength of solution used by Potts
is not given in his paper. Johnson discovered that strong solutions
are inimical to the nematodes and that dilution to as low as 0.15 per
cent. was necessary, this strength proving satisfactory for one series
of Rhabditis pellio. The writers have had a similar experience, finding
that strong solutions kill Cephalobus. dubius in a short time but that
very weak ones are quite efficient. In the work on which this paper is
based, 0.8-4.6 per cent. solutions were used. In fact, it was found un-
necessary to make up solutions of definite strength, except for special
Studies, since the stock cultures could be kept in flourishing condition
by occasionally (every four to six days) adding to the water in the mount-
ing-cell containing the nematodes the smal] amount of peptone which
would adhere to the point of a common dissecting needle. This method
of measure was, of course, subject to variation but within its limits the

152 WELCH AND WEHRLE

varying quantity seemed to produce no appreciable effect on the cultures.
and indicated a range of over five per cent. within which the solution
is suitable. The exact maximum and minimum dilutions for this species
were not determined. It should be mentioned in this connection that
even longer intervals between “feeding” were possible if necessity de-
manded. The writers had one peptone culture made up in the above-
described way which existed for over four weeks without renewal of the
food, the nematodes thriving and reproducing during the whole period.

As stated on a preceding page, Potts found that it was necessary
for peptone solutions to putrefy until a cloudy growth of bacteria had
developed and that it was “only in the presence of great numbers of
bacteria, or the substances formed by them, that the nematodes thrive
so well.”? In cultures of Cephalobus dubius maintained by the writers,
it was not necessary to develop any putrefaction of the peptone solution
but the dry peptone was added directly to the water of the rearing cell.
When new cultures were necessary, distilled water was placed in the
cell, a small amount of peptone on the end of a dissecting needle was
transferred to it, and the nematodes then introduced. This procedure
was followed a great many times and without any appreciable diminution
in the activities of the animals. Furthermore, cultures made up in this
way have been kept for a month without developing cloudiness and the
nematodes grew and reproduced rapidly. Since no attempt was made to
keep these cultures sterile, bacteria did develop in them to some extent
and it may be that in those cultures which remained perfectly clear a
limited amount of bacteria was present. It should be mentioned in
this connection, however, that a distinct cloudiness did appear in some
of the cyltures after standing several days, developing gradually into
a distinct brown color. This occurred rather commonly in summer and
was possibly due, in part at least, to the accumulation of bacteria. But,
with this particular nematode, the development of the brown color
was accompanied by a general deterioration of the culture and necessi-
tated either a removal and renewal of the greater part of the culture medi-
um or a transferrence of the nematodes to a new solution. It would
then seem that Cephalobus dubius requires only a very low development
of bacteria in the culture medium, if it is required at all, and that an
undue development of the cloudiness is usually detrimental.

REPRODUCTION IN CERTAIN NEMATODES 153

General Procedure

Certain general methods of procedure were found to be preferable,
and, in some cases, necessary to the maintenance of the cultures. In
stock cultures reared in glass mounting-cells, the optimum amount of
medium was found to be about two-thirds to three-fourths the capacity of
the cell. Most of the cells were kept approximately half filled. Smaller
amounts could be used but involved the danger of the cell drying up be-
tween observations.

Early in the work, it was noticed that when a stock culture was sealed
over completely with a vaselined cover-glass unfavorable conditions
became established and the culture would often die out. Experiments
were performed to determine the effect of the presence or absence of
an air-space above the medium and it was definitely demonstrated
that such a space must be provided, hence the above-mentioned proced-
ure of never filling the cells more than three-fourths full. Furthermore,
it was found that even with an air-space provided, unfavorable condi-
tions would develop in a few days if the cell was sealed air tight. This
situation was avoided by slipping the cover-glass back so that a small
crescent-shaped opening between the edge of the cover-glass and the
walls of the mounting-cell was produced. This method was entirely
satisfactory although it involved a certain loss of the water by evapora-
tion, thus necessitating an occasional restoration to the original level
in the culture-cell. The exact reason for this demand for ventilation is
not known. Johnson (’13, p. 612), who also discovered the necessity
for providing against completely closed cultures, thought that it was neces-
sary to permit the escape of gaseous decomposition products originating
in the culture medium. It is true that in cultures made from such
materials as yolk of hen’s egg, insect eggs, and ovaries of grasshoppers,
decomposition products were formed since they tend to quickly develop
offensive odors. On the other hand, Martin (’13, pp. 94-97; 143) has
pointed out the indispensability of free oxygen to the development of
the embryos of nematodes and it may be that the demand for oxygen
is the explanation. Since many of the culture media used were under-
going decomposition and since this process draws upon the available
oxygen, it is possible that the oxygen supply in one of the sealed culture-
cells, having either a small air-space or no air-space at all, would soon
be reduced to an unfavorable extent.

154 WELCH AND WEHRLE

Occasionally, even the partially closed stock cells, especially those
containing peptone, developed not only a cloudy appearance but also
a brown color which gradually increased in intensity. The specific
cause of this color is not known but it was evidently a result or an accom-
paniment of the general activity of organisms in the cell and almost in-
variably led to the death of the nematodes if the conditions were allowed
to continue. Thus it was necessary either to transfer nematodes to
an entirely new culture cell or else remove the old medium and replace
it with new. To accomplish the latter, the point of a finely drawn
pipette was thrust just below the surface of the medium in the stagnant
cell and the liquid very slowly removed. In this way, the eggs, young,
and adult nematodes which are always at the bottom were but little dis-
turbed and few of them lost in the process. Then the old medium was
replaced with distilled water and a small amount of peptone added.
If necessary, a number of the nematodes can be transferred to new cul-
ture-cells by means of the transferring needles already described. In
stocking a new culture, only a few nematodes need be transferred.

In studies of the reproduction, development, growth, and activities
of these nematodes, it was necessary to isolate individuals or eggs in
order to follow definitely and accurately the sequence of events. To
accomplish this end, cultures of a different type were used. The culture-
cell was made up in the same way as those for th: stock cultures except
that it was necessary to use a cell not over 5 mm. high. A single drop
of distilled water from a pipette was centrally placed in the bottom of a
clean, dry cell. This drop, if carefully placed, retained its integrity.
If the drop spread and came into contact with the edges of the cell, it
was discarded. To this drop of water was added a tiny bit of the food
material (the quantity which will just cling to the extreme point of a
dissecting needle) and the nematode or the egg transferred to it. Since
these single-drop cultures have a large air-space compared with the
bulk of the fluid and since they must be examined frequently in connec-
tion with the recording of data, these cells were kept tightly sealed and
in that way the danger of drying up was avoided. During the micro-
scopical examination, the whole cell was removed from the slide, thus
leaving the culture drop intact. It was necessary to guard against
undue evaporation during the exposure of the drop and all losses had to
be replaced. Transference of the nematodes, young or adult, was easily
accomplished by means of the needles already described. Eggs were

REPRODUCTION IN CERTAIN NEMATODES 155

~ transferred by the same means and likewise by the drawn out pipette
already described. The latter method was advantageous in transferring
a number of eggs at a time since they could be massed together in the
culture-cell and then drawn up in the pipette with a relatively small
amount of liquid. Furthermore, eggs were transferred by means of the
brush described in an earlier paragraph but this method was inferior
to the ones discussed above.

Starting Cultures from New Stock

Since Cephalobus dubius has been found but once and the entire
series of cultures is from the original stock, the writers have had little
experience in transferring this species from the conditions of nature to
those of the culture-cell. However, Diplogaster erivora has been brought
in from outside conditions and established in cultures a number of times.
New stock used by the writers usually came from grasshopper eggs and
was secured by breaking up the eggs in culture-cells, diluting the yolk
with distilled water, covering the cell in such a way as to leave an air-
space and a ventilating opening, and putting it aside under temperature
conditions of about 68-77° F. Often, after a few days, this nematode
would appear in the culture whence it could be transferred to a more
dilute medium of the same kind and then later to a different medium,
if desired. Mention has already been made of the fact that Merrill and
Ford (716) found this nematode parasitizing termites. Mr. Ford has
informed the senior writer that he could almost invariably secure a
culture by the following method: A number of termites are killed and
placed on the surface of water saturated soil in a small container. After
some hours, nematodes usually emerge from the heads of the termites
and continue their activities on the moist surfaces of the dead insects.
In a few days, the disintegrating bodies are often swarming with the
worms. ‘Then a number of them are removed to a culture-cell containing
the desired medium. Of those first transferred, the majority may die
but usually a few will survive the change and reproduce, starting a new
stock. Evidently, in a parasitic form, the transition from the body of
the termite to the conditions of a culture-cell is a severe one but the
immediate progeny of those which persist flourish in the new medium and
thereafter maintenance is simple. This mortality of the parasitic nema-
todes, when removed to cultures, confirms, in part, the experience of
Johnson (’13, p. 613) who frequently had great difficulty in starting cul-

156 WELCH AND WEHRLE

tures of Rhabditis pellio, although, when once started, the difficulties of
maintenance were considerably reduced.

Temperature Conditions of Maintenance

While no definite experiments have as yet been made to determine
the exact maximum and minimum temperatures for these nematodes,
certain observations are worthy of mention here. Temperature records
for rooms where some of the cultures were kept showed that both species
withstand successfully a considerable variation of temperature. In one
room, the daily maximum-minimum records sometimes showed a dif-
ference of as much as 33° F. Such variation usually occurred during
the winter months when the room temperature occasionally dropped from
74° to 42° F. Under these conditions, daily variation of 15-25° F. were
common and a minimum temperature of 40° F. for a limited time (4-6
hours) apparently had no serious effect on the cultures. In this same
room during the summer months, the variation was considerably less
but both maximum and minimum temperatures were much higher, e.g.,
92° and 84°F. It was found that when the room temperature rose much
above 80° F., cultures began to show signs of weakness.

In cultures of Cephalobus dubius, the nematodes began to die under
conditions of 90° F. These cultures were removed to an underground
concrete cave where an almost constant temperature of 80° F. was
maintained and evidence of strengthening was apparent. During the
following thirty days (July), when the temperature in the cave gradually
approached 87° F., the cultures showed increasing signs of weakness and
ultimately maintenance became difficult. In the following month, when
the temperature began to fall, signs of strengthening were evident as soon
as 80° F. was approached. When 78° F. was reached, the cultures were
soon flourishing again. In other respects, the conditions of rearing were
the same throughout this period. Evidently, the maximum temperature
for this species is near 90° F. and the optimum below 80° F. At the
present time, stock cultures are thriving under laboratory conditions
of 65-75° F.

Preliminary studies with reference to the influence of temperature
upon Diplogaster erivora were conducted by means of an air conditioning
machine having two large breeding chambers in which constant tem-
peratures (within very narrow limits) of 80° and 90° F., respectively,
were maintained. These nematodes could not be reared in the 90° F.

REPRODUCTION IN CERTAIN NEMATODES 157

chamber. Cultures submitted to that temperature soon showed decrease
of activity, suspended reproduction, and early death. Fresh cultures
reared in the 80° F. chamber and transferred to the 90° F. chamber
suffered the same fate. Nematodes reared in the 80° F. chamber also
exhibited some decrease in activity but continued to live and multiply,
producing four generations in forty-three days. Cultures, kept under
greenhouse conditions at a mean temperature of about 75° F., increased
more rapidly than did those within the 80° F. chamber and the rate of
development was greater, five generations being produced in forty-three
days. Still other cultures were maintained in the laboratory under a
variety of temperature conditions, and, according to the experience of
the writers, 65-75° F. is favorable for rearing Diplogaster @rivora.

PARTHENOGENESIS IN CEPHALOBUS DUBIUS MAUPAS

DISTRIBUTION

Cephalobus dubius is evidently a cosmopolitan species. Maupas
(00, pp. 555-556) states that it is very common in Algeria and that he
found it in a sample of red earth collected in the environs of Tananarivo,
Madagascar. In these localities, the nematode lives in rather poor earth
and is said to withstand long desiccation and to revive when the moist
conditions are re-established. Dr. N. A. Cobb states in a letter that it
occurs in various parts of the United States and is found in moist situa-
tions, feeding upon animal matter. He also states that on several occa-
sions it has been found on the surface of various insect eggs, and that the
young withstand desiccation.

SOURCE OF MATERIAL

The original stock for all of the writers’ cultures was secured accident-
ally in connection with the study of another nematode. In a prelimi-
nary experiment to determine whether Diplogaster erivora, a species
found feeding on the contents of grasshopper eggs, has the ability to
penetrate insect eggs or whether it depends upon some other agency
to provide the means of entrance, ordinary loam, sterilized by steam,
was placed in a jar. Grasshopper eggs, cleaned of all soil particles but
not subjected to special treatment, were introduced into this soil and
specimens of Diplogaster erivora in water added to the adjacent soil.
When examined later, the eggs were found broken and deteriorating.

158 WELCH AND WEHRLE

Under magnification, nematodes were seen in the egg contents and were
transferred to culture-cells. It soon became evident that they were
unlike Diplogaster erivora, and when submitted to Dr. N. A. Cobb for
identification they proved to be Cephalobus dubius Maupas. The original
source is thus in doubt, but since the soil in the jar had been sterilized,
it seems very probable that this nematode was carried into the culture
on the surface of the grasshopper eggs, either as an egg or in some sub-
sequent stage of development. Since there is evidence that at least
the immature individuals of this species can successfully withstand
desiccation, it is possible that they might have occurred on the surface
of the insect eggs in that state, resuming activity when introduced into
the moist conditions of the experiment.

PARTHENOGENESIS

Maupas (’00), in his extensive study of reproduction in nematodes,
found seven species in cultures of which he saw no trace of males. These
seven species, distributed among six different genera, included Cephalobus
dubius. He was inclined to admit the possible occasional occurrence
of males but states that if they do appear they must be very rare.

Since the beginning of this work, the writers have watched carefully
in the large number of successive generations for any appearance of males
but in vain. Mature individuals, isolated in culture-cells, always de-
posited eggs. Similarly, isolated eggs or young that attained maturity
always produced individuals which deposited eggs. Maupas expressed
uncertainty as to the constant absence of males because of the fact that,
in his studies, the greater part of the apparently parthenogenetic nema-
todes “se prétent mal 4 des cultures en grand” and that with one excep-
tion he was unable to examine very large numbers. That uncertainty
does not seem so significant since the writers were able, with their
methods of culture, to get this nematode in almost any quantity and
a very large number was examined for the possible appearance of males.
Since the nematodes were, in part, maintained under certain differences
and variations of food and temperature, it would seem that if males
ever do appear in this species they must be extremely rare and apparently
must develop under conditions of culture different in some unknown re-
spect from those of the writers. Cultures of this species are still under
observation in order that this matter, among others, may be more thor-
oughly tested. If it is later discovered that males do occasionally occur,

REPRODUCTION IN CERTAIN NEMATODES 159

it will be interesting to determine whether they have all of the activities
and functions of the sex or whether they resemble the imperfect males
which occasionally occur in several of the hermaphroditic nematodes.

As Lankester (’17, p. 504) points out, parthenogenesis signifies “an
exceptional and historically super-induced modification of the normal
process of sexual reproduction or gamogenesis in which the female gamete
or egg-cell does not unite with a male gamete or sperm-cell to form a
‘zygote,’ but proceeds to develop independently.” It is a well known
fact that protandric hermaphroditism (“Syngonism’’) is common among
the free-living nematodes and Cobb (15, p. 95; ’16, pp. 198-199) suggests
the desirability of re-examining the supposedly parthenogenetic forms
to determine whether some of them are not actually “‘syngonic.” This
suggestion was made on the basis of a study of a series of syngonic free-
living nematodes in which the spermatozoa present were smaller and
smaller until they reached the optical limits of present instruments and
he finds himself unable to assert the non-existence of spermatozoa in
certain nematodes merely because he has not succeeded in finding them.
This suggests that since both the parthenogenetic and the hermaphrodi-
tic nematodes have the unmodified form of the female, the former may,
at least in some cases, possibly be instances of masked hermaphroditism.

The writers have not thus far made any microscopical examination
of the gonads to determine the above-mentioned point and no comment
can be made upon it except that the complete absence of males in con-
tinuous cultures maintained for over three years seems to indicate the
parthenogenetic method of reproduction. If the animal were herma-
phroditic, it would necessitate that every egg produced be fertilized and
that fertilized eggs invariably produced hermaphrodites in order for the
results of the writers’ cultures to have been possible. Furthermore,
as Maupas and others have pointed out, the hermaphroditic forms show
an unbalanced relation between the number of spermatozoa and ova
produced and the unfertilized ova do not develop but disintegrate after
they are laid. In the case of Cephalobus dubius, all of the eggs laid were
capable of development. Also, in the majority, if not in all hermaphrodi-
tic nematodes, males appear even though they may in some species be
rare and imperfect in their sexual functions. At present, there seem
to be no grounds for considering Cephalobus dubius other than a par-
thenogenetic form.

160 WELCH AND WEHRLE

Ovi position

In order to obtain data on the reproductive capacity of the females,
oviposition records were made as follows: Eggs, isolated from vigorous
stock cultures, were placed individually in a single drop of distilled water
in a glass mounting-cell. Frequent observations were made and the
time of hatching carefully noted. A trace of yolk of hen’s egg was then
added and daily observations continued until the death of each individual.
When oviposition began, the number of eggs appearing in the cell each
day was recorded and the female transferred to a new culture, thus avoid-
ing the labor of removing the eggs from the cell.

Cultures of thirty-six different individuals, representing a number of
successive generations, were carried through the life cycle to cessation
of oviposition and subsequent death and showed considerable variation
in the length of the egg-laying period. These cultures were all main-
tained under the same conditions with respect to food but the tempera-
ture was that of the laboratory and subject to an average daily variation
of about 18° F., the extremes being approximately as follows: minimum,
40-58° F.; maximum, 66-76° F. Temperature was the only factor which
varied to any extent and the precise influence of this factor was not de-
termined. Under these conditions, the duration of the egg-laying period
was found to have a variation of 6—44 days, the average being about 16 days.
This variation seems surprisingly large and leads to the suspicion that those
nematodes with a short egg-laying period are individuals which, for some
reason, had their normal existence shortened. But, since no account
was taken of those individual life histories which were ended at a time
when the egg production rate was high and only those which showed
an acceleration followed by a subsequent retardation were considered,
it does not appear that all such instances can be so interpreted. It
should be stated that the maximum oviposition period of 44 days given
in this set of records is much higher than any of the others, the next
lowest being 31 days.

Egg-laying, when once initiated, continues uninterruptedly until its
decline at the end of the life of the individual. None of the records
showed any evidence of definite periods of cessation of egg-laying followed
by active resumption of oviposition. Only two instances of 24-48 hour
periods with no oviposition appears in the records and these occur at
the end of the life of the nematode. The eggs are laid singly and at a

REPRODUCTION IN CERTAIN NEMATODES 161

varying rate depending upon the age of the parent. The most charac-
teristic feature of the records on all individuals presenting any evidence
at all of a normal life cycle is the rather gradual increase in the daily
rate of egg-laying during the first part of the oviposition period and the
more or less gradual diminution in the last part. In every case, the maxi-
mum daily number of eggs deposited appears well within the oviposition
period, ordinarily near the middle. Usually the initial and concluding
daily rates are very slow, the latter often ceasing completely before the
death of the nematode. In a few individuals only was the initial daily
record above 10. The increase and decrease of the daily rate were not
regular for any of the individuals studied. The maximum production
of 27 eggs in one day occurs in the record of nematode No. 36. <A few
other records are close rivals. According to Maupas (’00, p. 560), “ Par
une température de 20° c., le maximum d’ceufs pondus, dans les vingt-
quatre heures, est de 12 4 13.”’ It is evident that the daily rate of egg
production is, at least at times, much higher than Maupas’ record would
indicate.

The total number of eggs per individual varied from 33 to 285, the
average being 139. In general, those nematodes which lived longest
and thereby had the greatest oviposition period produced the largest
number of eggs but, in the writers’ records, no constant relation exists
between the duration of the oviposition period and the total number
of eggs. For instance, nematode No. 55 produced 101 eggs in 9 days but
nematode No. 79 deposited only 77 eggs in 13 days; nematode No. 24
produced 270 eggs in 44 days but nematode No. 85 produced 285 eggs in
30 days. Maupas (’00, pp. 561-562) reported a maximum of 415 eggs
deposited over an egg-laying period of about 4 months.

In addition to the above, another set of oviposition records were made
from cultures kept in an underground, concrete cave, where, during the
period involved, the daily temperature variation was within 5° F., usually
within 4°F. The average maximum temperature was 77.8° F., the varia-
tion being 75-79° F., and the average minimum temperature was 73° F.,
the variation being 71-76° F. While the temperature was not com-
pletely controlled, the fluctuation was small and the results are of interest
when compared with those already described. The remaining conditions
of the cultures were like those of the other series with the exception that
peptone was used instead of the yolk of hen’s egg. Six individuals were

162 WELCH AND WEHRLE

successfully carried through their complete life cycle and the egg-laying
period varied from 15 to 39 days. The total number of eggs deposited
by each individual varied from 78 to 234. This series of cultures also
presented the characteristic increase and diminution of the daily rate
of oviposition which has already been described. The lack of a definite
relation between the length of the egg-laying period and the total number
of eggs deposited was also apparent although the general tendency for
the longer periods to be accompanied by the larger number of eggs was
evident. Owing to the difference in the numbers of individuals studied
in the two series, a more detailed comparison is not possible.

After oviposition, the development of the embryo goes on rapidly
and within a few hours the egg, under magnification, shows signs of
internal activity. Records on the development of 88 uggs from deposi-
tion to hatching showed 3.3 days as the average egg period, the variation
being 2.5-4 days. This is in close agreement with Maupas’ statement
(00, p. 561) “les ceufs mettent trois jours 4 parcourir leur embryogénie
jusqu’a éclosion.”

Immature Period

Length of Larval Stage——In hen’s egg cultures kept under the con-
ditions of the laboratory, the larval period of 48 individuals of different
progenies showed a variation of 8-17 days, although the usual variation
was 8-14, 17 occurring but once and 14 being the next lowest number. —
The average larval period, based upon the above-mentioned records, is
10.1 days. In this paper, the larval period is regarded as extending
from the moment of hatching to the deposition of the first egg, and not
to the cessation of growth as will be discussed later. The appearance of
the genital pore is an indication of maturity or closely approaching
maturity but it was thought best to use the first oviposition as the final
limit of the immature stage. Maupas (’00, p. 561) reported the larval
period to be 10-11 days, at a temperature of 68° F. He also used the
appearance of the first egg as evidence of the completion of larval
existence.

In peptone cultures maintained under the more uniform temperature
conditions of the underground cave described previously, the larval
period of 10 individuals had a variation of 8-14 days, the average being
10.8 days. It will be noted that this agrees closely with the cultures
reared under laboratory conditions and it appears that limited differences
of temperature and the comparative culture values of peptone and yolk

REPRODUCTION IN CERTAIN NEMATODES 163

of hen’s egg had no striking effect on the time interval demanded for the
attainment of sexual maturity.

Rate of Growth—Careful, daily measurements of length of 8 indi-
viduals reared in peptone cultures under the cave conditions were made
for about 22 days, a period of time well beyond maturity and the cessa-
tion of growth. It was noted that the period of growth and the Jarval
period are not coterminal, but that growth usually continue. 3-6 days
after the production of the first egg. During this time, the increase in
body-length has a variation of 0.019-0.076 mm., the average being
0.0527 mm.—about 10 per cent. of the average body-length of the com-
pletely formed individual.

At hatching, the larva is only 0.140-0.179 mm. long, the average of
16 specimens being 0.162 mm., and, from that time to the attainment of
the full-grown condition, growth is continuous and approximately uni-
form. The growth curves of different individuals are very similar. The
average daily increase in length was found to be 0.0258 mm., the varia-
tion being 0.019-0.03 mm. There is no evidence of a definite maximum
daily rate of growth anywhere in the period and apparently growth,
when completed, ceases almost as abruptly as it begins. The average
growth period was approximately 16.3 days, with a variation of 14-18
days.

Length of Life

The complete life showed a variation of 20-61 days. Only 1 nematode
lived 61 days, but 8 of 29 individuals lived 40-50 days, and 11 lived 30-40
days. The variation indicated above appears to be rather large and
may have been due to some peculiar combination of factors in the dif-
ferent cultures, although all were given the same kind of food in approxi-
mately the same amount and kept under the conditions of the laboratory.
Maupas (00, pp. 561-562) records one individual which lived for approxi-
mately five months. The egg-laying period has already been discussed
and it thus appears that if the first eggs of the parent be considered, a
new generation can be produced in the cultures every 11-19 days, or about
two generations per month.

REPRODUCTION IN DIPLOGASTER ZZRIVORA COBB
SOURCE OF MATERIAL
This nematode was first discovered in 1914 infesting the eggs of
grasshoppers after they had been deposited in the ground. Specimens.

164 WELCH AND WEHRLE

were sent to Dr. N. A. Cobb who reported them as representing a new
species. Later, Merrill and Ford (’16) found nematodes infesting the
termite, Leucotermes lucifugus, which, when submitted to Cobb, proved
to be the same species as the one found in grasshopper eggs and he
described it under the name Diplogaster erivora. Merrill and Ford made
a special study of this nematode in relation to the termite host and pub-
lished, in addition to Cobb’s original description of the species, certain
data on the reproduction and habits of this worm as observed in cultures.
The present account includes the results of a more prolonged study of
certain features of the reproduction, based upon continuous culture
studies of more than three years.

OVIPARITY

Merrill and Ford (’16) have already pointed out the fact that males
and females are continually present and that the males are functional.
Cobb, in the same paper, described the morphological features of the two
sexes. The writers have also found this species reproducing exclusively
by the bisexual method. No evidence of hermaphroditism or partheno-
genesis appeared, although evidence of such phenomena was sought con-
tinually

Copulation

Copulation was easily studied in the cultures and the observations
of the writers essentially confirm the brief account of Merrill and Ford
(16, pp. 125-126). Increased activity on the part of the male before
mating was noticed. A female will copulate with several different males
in a short time and the same is true of the behavior of the male towards
different females. As will be shown later, observations indicate that a
female must mate two or more times in order to produce fertile eggs
throughout the adult life.

Fertile Eggs

The normal, fertile egg of Diplogaster erivora is oblong, the average
dimensions from forty-five measurements being 0.064 mm. and 0.035
mm., the variation being within 0.007 mm. ‘The outer covering consists
of a tough, membranous coat. Eggs are always deposited singly.

Infertile Eggs
An egg, which has seemingly never been fertilized and from which a
nematode never develops, is designated as an infertile egg in these

REPRODUCTION IN CERTAIN NEMATODES 165

studies. Such eggs are larger than the fertile ones, their dimensions being
about 0.084 mm. and 0.043 mm. They are slightly variable in shape
but are usually suboval. The envelope consists of a very thin membrane
and encloses the finely granular, light colored contents. The general
appearance of such eggs is sufficiently different from that of the fertile
eggs to make it comparatively easy, with a little practice, to recognize
them at sight in the cultures.

Relation of Copulation to Egg Production

An interesting relation between copulation and the production of both
fertile and infertile eggs exists in this species. Fertile eggs were never
deposited by a female previous to the initial copulation. A female, upon
being mated, will, for a time, lay fertile eggs after which infertile ones
will be deposited until another mating occurs. Upon being mated the
second time, the female will again produce fertile eggs. For example,
female No. 12 was mated at maturity, after which the male was removed
from the cell. Twelve fertile eggs were deposited by this individual
during the next 48 hours. During the following 60 hours, 8 egg- were
extruded, all of which were infertile. After 6 days, this female was
mated a second time and as a result, within 3 hours after copulation,
fertile eggs were again deposited. Fifteen fertile eggs were laid during
the following 3 days and 2 fertile eggs were found in the body of the
female at death. Essentially, the same results were obtained in several
different individuals maintained under similar conditions. On the other
hand, female No. 88 was kept constantly exposed to males throughout
her mature life and fertile eggs were uninterruptedly deposited until her
decease. After the first mating of this female, eggs soon appeared and
the number rapidly increased until well towards the end of the 2nd day
at which time the maximum deposition of 19 eggs occurred. From this
time on, there was a gradual reduction in the number of eggs produced
until cessation occurred at the end of the 6th day, death ensuing about
24 hours later.

Occasionally, a female would completely exhaust her ability to deposit
fertile eggs before any infertile ones were produced but, in the majority
of cases, the approaching cessation of fertile egg production was indicated
not only by the reduction in the number but also in the appearance of a
mixture of fertile and infertile eggs leading to the final disappearance of
the former. After a second or subsequent mating, the same phenomenon

166 WELCH AND WEHRLE

usually occurred except in the reverse order, although, in some cases,
there was an abrupt cessation of the infertile egg production after re-
mating the female. Commonly, the maximum‘ egg production follows
the first mating, subsequent ones being followed by the deposition of a
smaller number of eggs over a shorter length of time, but this is not a
constant feature since a few exceptions appear in the records, as for
example, female No. 81 was mated 3 times within a period of 11 days
and the maximum production followed the third mating.

It thus appears that in order for the female of Diplogaster erivora to
attain her complete reproductive capacity, she must be mated at iatervals
throughout the egg producing period. One mating is insufficient but
the reasons for this insufficiency are not definitely known at present. In
all of the matings, the period of production of fertile eggs never exceeded
3 days and it may be that this represents the extent of the life of the
spermatozoa after they have been transferred to the female. There is
no reason for believing that the fertilization is other than /ysterogamic
(Lankester, ’17, p. 505) and possibly fertile eggs cease to appear when
all the spermatozoa of a single mating have been exhausted, but, if this
be the case, there must be considerable variation in the number of sper-
matozoa transferred to the female at copulation since, according to the
writers’ records, the number of fertile eggs resulting from a single mating
varied widely, as for example, initial matings resulted in 7-113 fertile
eggs. Possibly the length of copulation, which is known to vary in this
species, determines, in part at least, the number of male cells transferred.

Maupas (00, pp. 586-587; 601-602) pointed out that there is a striking
imperfection in the protandric hermaphroditic nematodes in which fertile
eggs will be produced until the supply of spermatozoa is exhausted and
then the same nematode will continue to produce infertile eggs in num-
bers 2 or 3 times greater than the fertile ones. The evidence seems to
support the contention that this condition is the result of the failure of
the ovo-testis to develop enough male cells. The infertile eggs invariably
deteriorate. He also found that the occasional males of these herma-
phroditic species sometimes fertilized the hermaphroditic individual
after it had exhausted its own supply of spermatozoa. These cases
may be comparable, if not homologous, to the condition in Diplogaster
erivora. Certainly, the disadvantage to the species is similar. Since
all of the infertile eggs of Diplogaster erivora deteriorate, the reproductive
capacity of a single female is limited greatly unless males are ever present
to fecundate her.

REPRODUCTION IN CERTAIN NEMATODES 167

VIVIPARITY

At times, another form of reproduction occurs in the life history of
this nematode in which living young appear within the body of the moth-
er. This phenomenon existed many times in the cultures and was care-
fully studied. The first indication of this change in the normal repro-
ductive procedure is the appearance of one or more very tiny nematodes
within the body of the parent and inside of the uterus. These young
move about actively and increase in size at a rapid rate. In time, they
break their way through the wall of the uterus into the body-cavity of
the mother and begin to actively attack her internal organs, soon causing
her death. The young continue to feed upon the body contents of the
dead parent until, in many cases, the whole interior is hollowed out,
leaving nothing but the transparent cuticula within which the developing
young wriggle about. From this empty parental cuticula, the young
escape to the exterior and take up an independent existence in the sur-
rounding nutrient medium. Approximately one-third to two-fifths of
the complete growth may be attained within the parent. Merrill and
Ford (716, p. 126) observed this phenomenon in the parasitic strain
of this species which they studied and they state that “Usually they were
unable to escape, although instances were observed where they escaped
through the genital pore of the mother.” They also figure a dead female
containing 14 young, all of about the same degree of development. It
might be inferred from the above quotation that few of these young are
able to complete their development, but the long observations on which
this paper is based show that, in the strain from the grasshopper eggs,
not only is the phenomenon fairly common but that the large majority
of the young so developed escapes through the genital opening of the
mother or through some rupture of her body-wall. Not only do such
young complete their growth but they are perfect individuals and capable
of reproduction. The number of young developing within the parent
is variable. The writers have records of as many as 20 appearing at about
the same time and also some evidence that even more may be so produced.
On the other hand, the number may be as low as 3 or 4. These larve
develop into both males and females and, since living young were never
observed in females which had not at some time been exposed to males,

it appears that they arise from fertilized ova and not from parthenogene-
tic ones.

168 WELCH AND WEHRLE

Reproduction of this viviparous sort has been observed before in
nematodes. Maupas (’00) found it to be a common occurrence in
Rhabditis elegans, Rhabditis caussaneli, and Diplogaster robustus in which
the eggs are not deposited as rapidly as they arrive in the uterus but tend
to accumulate there, the delay causing them to be deposited ultimately
in an advanced stage of development. Some of the young hatch in the
uterus and are expelled along with the unhatched eggs but when the sup-
ply of spermatozoa is exhausted and the infertile eggs pass into the uterus,
the young hatch, feed upon the infertile eggs accumulating there, grow,
rupture the wall of the uterus, scatter in the general cavity of the mother,
disorganize and devour the internal parts, and ultimately escape to
the exterior. Pérez (Conte, ’00b, p. 375) observed viviparity in Rhabditis
teres, Conte (’00a; ’00b) recorded it in Rhabditis monohystera and Diplo-
gaster longicauda, and Southern (’09, pp. 93-94) described the occasional
appearance of young within the body of the mother in Rhabditis brassice.
Merrill and Ford (’16, p. 120) apparently found a similar occurrence in
Diplogaster labiata in which “Occasionally a young nematode hatched
within the body of a dead female,” but no statement is made as to the
ultimate fate of such individuals.

The factors initiating and influencing the appearance of this vivi-
parity are not definitely understood. Conte (00a) found that Rhabditis
monohystera “vivipare sur colle de pate, est ovipare sur peptone” and that
the conditions of development are influenced by the nature of the nutri-
tive medium. In another paper, Conte (’00b) points out that Maupas
(99) attributes the appearance of this viviparity to two causes, inanition
and senility. Conte, however, found evidence that putrefaction in
the medium was a cause, at least in the case of Rhabditis monohystera.
“Tout en admettant avec lui que, dans certaines espéces, l’inanition et
la sénilité aménent le parasitisme embryonnaire, je crois que ce phéno-
méne peut étre provoqué par d’autres causes et notamment, ches Rhab-
ditis monohystera, pax la putréfaction du milieu. D’une fagon générale,
je pense qu’il est en relation avec un état morbide de la mére.” In
connection with his studies of the production of eggs or young in these
nematodes, he finds it possible to distinguish the following stages which
are closely related to the character of the nutritition:

Absolute oviparity—deposition of unsegmented eggs.
Relative oviparity—deposition of eggs undergoing segmentation.

' REPRODUCTION IN CERTAIN NEMATODES 169

Ovo-viviparity—deposition of gegs containing active embryos.

Viviparity—deposition of young which hatched in the uterus of the

mother.

Embryonic parasitism—consumption of morbid mother by her off-

spring.

There is certainly a tendency in Diplogaster erivora for this form of
reproduction to appear towards the end of the reproductive period and
while the parent may live and show body movements for a brief period
after young appear within her body, her reduced vitality is apparent
and is an accompanying feature, if not the causative one, of this phenom-
enon. However, the writers have evidence that age may not always
be an accompanying factor, in fact, there is circumstantial evidence that
any set of conditions which interferes seriously with the well-being of
the female may lead to the appearance of living young within her body,
even early in the reproductive period. It is therefore the opinion of the
writers that this viviparity is the result of reduced vitality of the mother
rendering her incapable of discharging the eggs. It also appears that
eggs formed early in the reproductive period may hatch within the body
as well as those produced at the end, and the writers have thus far dis-
covered no positive evidence of any inherent predisposition of the last
formed eggs for internal hatching.

There is no question that this appearance of young within the body
of the mother constitutes a form of reproduction and that the resulting
offspring are just as capable of continuing the species as those arising
from hatching outside of the body. In reality, these two forms of repro-
duction are only superficially distinct and the use of the terms oviparous,
ovo-viviparous, and viviparous is mainly one of convenience rather than
one of exact distinction. Lankester (’17, p. 505) holds that “really all
animals are viviparous, for the birth-product is a living thing whether
it is a naked egg-cell or more or less advanced in development. The
enclosure of the birth-product is a shell or case, which has given rise to
the term ‘oviparous’ is not of any value as indicating the real degree of
development of the young at birth, for in some cases unfertilized egg-
cells, in others mere discs of developing embryonic cells (as in birds, etc.),
and in yet other cases well-shaped young ranging from the early larva
of Some invertebrates up to the completely formed miniature of the adult,
as In some of the shell-bearing snails, may be enclosed within an egg shell
when ‘laid’ by the mother. There is accordingly no general importance

170 WELCH AND WEHRLE

to be attached to the distinction between ‘viviparous’ and ‘oviparous’
animals.” In this paper, these terms have been retained for the sake
of convenience.

PROPORTION OF SEXES

In the cultures studied by the writers, the total egg production of
females varied widely, the average being about 55 eggs per female. The
maximum observed was 196. In an attempt to determine the propor-
tion of the sexes and the number of eggs and young per female, eggs
selected at random were isolated, each in a separate culture. Females
resulting from these eggs were mated at regular intervals throughout
their lives. A complete daily record was kept of all eggs laid by these
females and the sex of the resulting young determined by rearing them
to maturity. Twenty-two females were studied in this way and, of the
437 resulting offspring, 182 developed into males and 291 became females.
Since it was not possible to eliminate a certain mortality among the eggs
and larvze, the numbers given above can be regarded only as a general
indication. They do show, however, that, in contrast to some of the
bisexual species, the males are very common although the females are in
the majority. These results agree, in general, with those of Merrill and
Ford (’16, p. 126).

Records from the progeny of 22 females show that in the vast majority
of cases both males and females appear in each progeny and also that in
most of the progenies, females were numerically dominant. Since all
the evidence indicates that the species is exclusively bisexual in its mode
of reproduction, a sufficient number of males is demanded to maintain
the generative processes, but since it was observed that a single male
may and often does copulate with a considerable number of females, it
seems probable that the smaller number of males indicates no important
disadvantage in the multiplication of the species. In fact, the numerical
dominance of the females coupled with fewer but sexually active males
may possibly facilitate the production of a larger number of offspring,
even if the locomotor ability is poorly developed.

Cobb (18, p. 477), in discussing the comparative rarity of males
in free-living nematodes, states that ‘“There is reason to think that in
some of the species the males are short-lived, and that this is the reason
they are so rarely seen. The males are often so much smaller than the
females that they are easily overlooked, or mistaken for young, so that in

REPRODUCTION IN CERTAIN NEMATODES 171

such cases the rarity of the males may easily be overestimated.” Since
the length of life of the males and females of Diplogaster erivora is vir-
tually the same and since the length of the female exceeds the length of
the male by only about one-fifth, it would seem that these features have
not affected the observations on this species.

RATE OF GROWTH

The young nematode, upon emerging from the egg, is active and moves
about in much the same way as the adult. Its average length at emer-
gence is about 0.238 mm. and the average diameter about 0.015 mm. A
number of specimens were carefully studied in individual cultures for
the rate of growth, all being reared under the same conditions and all
living 12-23 days after hatching. Careful measurements were made at
the time of emergence from the egg and at regular intervals of 24 hours
thereafter throughout the life of each individual. Data were secured
from 4 males and 5 females which were carried through their entire
existence, each manifesting all of the activities of a normal individual
and living for some time after growth had ceased. From the sedata,
growth curves were constructed for the increase in length and diameter.
These curves showed a striking similarity, not only in the different indi-
viduals of the same sex, but also in the individuals of the different sexes.
Furthermore, the curves for the increase in length and the increase in
diameter showed very close correspondence in every case. Growth
begins immediately at hatching and continues uninterruptedly for a
period, after which it ceases permanently. Composite graphs con-
structed on the basis of the individual growth curves showed that growth
in both length and diameter ceases, on the average, on the 8th-9th day
after hatching. In all cases except one, the variation from this average
was very slight, the exception showing no growth after the 4th day. The
average length of the males at the end of the growth period was 0.864 mm.
and the diameter 0.052 mm., while the length of the females was 1.105
mm. and the diameter 0.068 mm. The length of life of the adult follow-
ing cessation of growth varied with the individual from 6 to 15 days
inclusive.

LENGTH OF LIFE

The length of the life of this nematode is, no doubt, subject to varia-
tion under different conditions. One series of eggs kept under average

172 WELCH AND WEHRLE

temperature conditions of about 75° F., variation within 7°, required
an average of 17.9 hours from the time of oviposition to hatching,
the variation being 17-20 hours. A series of 29 individuals showed an
average larval period of 3.75 days, the variation being 1.3-8 days.
Twenty-three individuals of the same series had an average adult life
of 13 days, the variation being 5-21 days. The average life of the
individual outside of the egg was found to be about 17.1 days. Including
the egg stage, the average life was approximately 18 days. Assuming
copulation on reaching maturity, a generation can be secured in about
4.5-5 days. But very little difference was observed in the length of life
of males and females.

SUMMARY

METHODS

1. Some of the free-living and semi-parasitic nematodes can be reared
generation after generation in artificial media and their study thus
facilitated. Two species, Cephalobus dubius Maupas and Diplogaster
erivora Cobb, were cultured continuously for over three years.

2. Cylindrical glass mounting-cells sealed with pure vaseline to
ordinary microscope slides and covered with vaselined cover-glasses
were found to be the most suitable containers for cultures. Methods
of transference are described.

3. Of the numerous substances used as media, a very dilute solution
of peptone, diluted yolk of hen’s egg, and Pfeffer’s synthetic agar were
most extensively used. The eggs, ovaries, and body tissues of a number
of insects were found to be favorable when properly prepared.

4. Unfavorable cultural conditions developed in stock cultures which
were tightly sealed. Provision for ventilation was necessitated.

5. In starting cultures of Diplogaster erivora from new stock taken
from nature, mortality in the first generation was high but usually a few
survived in the new medium and subsequent maintenance then became
simple.

6. Temperatures above 80° F. are unfavorable, 90° F. and above
proving fatal. These nematodes withstand a considerable fluctuation
of temperature, e.g., 32° F. in 24 hours. Temperatures as low as 40° F.
can be withstood, at least for a limited time. Optimum temperature
conditions seem to be near 65-75° F.

REPRODUCTION IN CERTAIN NEMATODES 173

CEPHALOBUS DUBIUS

1. Observations on long continued cultures of Cephalobus dubius
involving many individuals and generations revealed no traces of males
and reproduction seems undoubtedly parthenogenetic. If males ever
appear, they must be extremely rare and develop under conditions of
culture different in some unknown respect from those employed by the
writers.

2. The egg-laying period has a variation of 6-44 days, average about
16 days. Oviposition, once initiated, continues uninterruptedly. The
daily rate increases somewhat gradually to the middle of the period and
then declines. As many as 27 eggs per day may be deposited. The
total number of eggs per individual showed a variation of 33-285, average
139. Apparently, all eggs were capable of normal development.

3. Under cultural conditions, larve emerge from eggs 2.5-4 days
after oviposition and reach maturity in 8-14 days.

4. The growth period and the larval period are not coterminal, growth
usually ceasing 3-6 days after sexual maturity, during which time an
increase in body-size of about 10 per cent. occurs. Larval growth is
continuous and approximately uniform. The average daily increase
in body-length is about 0.026 mm. Growth curves of different individ-
uals are very similar.

5. The length of life has a variation of 20-61 days. Computing from
the first eggs of a parent, a new generation can be secured in cultures
every 11-19 days.

DIPLOGASTER ZRIVORA

1. Diplogaster erivora is bisexual and males are completely functional.
One female may copulate with several males and males may behave simi-
larly towards different females.

2. Both fertile and infertile eggs are deposited. Fertile eggs follow
mating for a time, after which infertile eggs are laid until a second mating,
after which the same sequence usually occurs. Constant exposure to
males may completely prevent deposition of infertile eggs. Approaching
cessation of fertile egg production is often indicated by the appearance
of a mixture of fertile and infertile eggs. Usually, maximum oviposition
follows the first mating.

3. All of the evidence indicates that the female must be mated at
intervals throughout the egg producing period in order to fulfil her com-

174 WELCH AND WEHRLE

plete reproductive capacity. The insufficiency of one mating is not
definitely understood but it seems probable that infertile eggs appear
upon the exhaustion of the spermatozoa received from a single mating.
A similar imperfection in the reproductive ability appears in the pro-
tandric hermaphroditic nematodes and is explained by Maupas and others
as due to exhaustion of the limited supply of spermatozoa.

4. Viviparity occurs from time to time. Young appear, first within
the uterus, then later within the body-cavity of the mother, feeding upon
her internal organs, untimately causing her death. In many cases, the
interior is completely consumed, leaving nothing but the transparent
parental cuticula from which the young escape to take up independent
existence in the surrounding medium. Young so produced evidently
originate from fertilized eggs and develop into both males and females
capable of normal reproduction.

5. This form of viviparity has been observed by other workers and
certain factors have been proposed as being responsible for the appearance
of this phenomenon, namely, inanition and senility (Maupas), and putre-
faction of the medium (Conte). The writers have incomplete evidence
that it is the result of any factor or group of factors which reduce the
vitality of the mother. Definite evidence was secured to show that,
at least in Diplogasier erivora, age is not always an accompanying feature
but viviparity may appear at any time during the egg-laying period.

6. Females are numerically dominant, both in mass populations and
in single progenies. Males, however, are common and very rarely
absent, even in a single generation. Although fertilization is evidently
necessary for any reproduction, it is probable that the smaller number
of males is compensated for by their constant presence and their ability
to copulate with several females.

7. Growth curves based upon daily measurements of length and
diameter are strikingly similar for all individuals, irrespective of sex.
Composite graphs showed the cessation of growth occurred on the 8th-
9th day. The length of life following cessation of growth has a variation
of 6-15 days..

8. The average length of life under cultural conditions is about 18
days. A new generation can be secured every 4.5-5 days. No marked
difference in the length of life of males and females was observed.

REPRODUCTION IN CERTAIN NEMATODES 175

LITERATURE CITED
Byars, L. P.
1914. Preliminary Notes on the Cultivation of the Plant Parasitic Nematode,
Heterodera radicicola.
Phytopathology, 4:323-327.
Cobb, N. A.
1915. Proceedings of the Helminthological Society of Washington.
Journ. Parasit., 2:93-95.
1916. Proceedings of the Helminthological Society of Washington.
Journ. Parasit., 2:195-200.
1918. Free-living Nematodes. Fresh-water Biology, by Ward, H. B., and
Whipple, G. G., pp. 459-505.

Conte, M. A.
1900a. De J’influence du milieu nutritif sur le développement des Nématodes
libres.

C. R. Soc. Biol., 52:374-375.
1900b. Sur les conditions de ponte des Nématodes.
C. R. Soc. Biol., 52:375-376.
Johnson, G. E.
1913. On the Nematodes of the Common Earthworm.
Quart. Journ. Micr. Sci., 58:605-652.
Lankester, E. R.
1917. The Terminology of Parthenogenesis.
Nature, 99:504-505.
Martin, A.
1913. Recherches sur les conditions du développement embryonnaire des Néma-
todes parasites.
Ann. Sci. Nat., Zool., (9), 18:1-151.
Maupas, E.
1899. La mue et l’enkystement ches les Nématodes.
Arch. Zool. exp., (3), 7:563-628. 3 pls.
1900. Modes et formes de reproduction des Nématodes.
Arch. Zool. exp., (3), 8:463-624. 11 pls.
Merrill, J. H., and Ford, A. L.
1916. Life History and Habits of Two New Nematodes Parasitic on Insects.
Journ. Agr. Research, 6:115-127.
Metcalf, H.
1903. Cultural Studies of a Nematode Associated with Plant Decay.
Trans. Am. Micr. Soc., 24:89-102.
Oliver, W. W.
1912. The Cultivation of an Ectoparasitic Nematode of a Guinea Pig on Bac-
teriologic Media.
Science, (n.s.), 36:800-801.

176 WELCH AND WEHRLE

Osborn, H.
1898. The Hessian Fly in the United States.
Division of Entomology, U. S. Dept. Agr., Bull. 16,n. ser. S8pp. 2 pls.
Potts, F. A.
1910. Notes on the Free-living Nematodes.
Quart. Journ. Micr. Sci., 55:433-484.
Southern, R.
1909. On the Anatomy and Life-history of Rhabditis brassice, n. sp.
Journ. Econ. Biol., 4:91-95. 1 pl.

A NEW DIATOM-EATING FLAGELLATE 177

A NEW AND REMARKABLE DIATOM-EATING FLAGELLATE,
JENNINGSIA DIATOMOPHAGA NOV. GEN., NOV. SPEC.

AsA. A. SCHAEFFER

Diagnosis. Shape, cylindrical, 180 microns long by 40 microns in diameter;
very metabolic. Flagellum, large, 150 microns long. Cuticula with spiral striations
1 to 2 microns apart; numerous movable club shaped appendages about one and one-
half microns long on the striations. Large central nucleus, 35 microns in diameter.
Large contractile vacuole near the anterior end. Several rod-like structures in the
pharynx immediately internal to a large mouth at the anterior end. Numerous bodies
in the form of rings or strongly biconcave discs from 1 to 3 microns in diameter, in the
endoplasm; large clear spheres up to 6 microns in diameter sometimes present. Loco-
motion, creeping. Food, exclusively diatoms. Reproduction, asexually, by longi-
tudinal fission. Habitat, marshes, among algae and diatoms.

This remarkable flagellate has come under my observation on three
different occasions: February 27 and March 31, 1916, and March 5, 1918.
In the three instances, material was collected from the Lonsdale marshes
near Knoxville, and allowed to stand in vessels in the laboratory for
several months. The organism may be considered rare, for frequent
examinations of material from these marshes during the past five or six
years has revealed its presence only three times.

This flagellate does not occur in large numbers, not more than a few
hundred being found at any one time. And their length of life in the
culture is as short as their number is small. In none of the three cases
mentioned did they remain in the culture for more than a week. Its
rarity, and the difficulty with which it may be cultured are unfortunate
from the point of view of the biologist, for its predatory animal nature
stands out in striking contrast to the plant-like character of some of its
immediate relatives.

I propose for this organism the generic name Jenningsia, in honor
of my friend Professor H. S. Jennings, and the specific name diatom-
ophaga.

Jenningsia is one of the largest members of the flagellates. Although
the average length is about 180 microns, several individuals were found
which measured 260 microns, exclusive of the flagellum, which in these
individuals measured 170 microns, and in the smaller individuals, 150
microns. The shape of the body is cylindrical, with slightly tapering
anterior end and very blunt but also slightly tapering posterior end. The

178 A. A. SCHAEFFER

flagellum is very stout and during locomotion is directed straight ahead,
only the apical part being usually in active motion (figs. 1, 2).

In the general character of its movements this organism resembles
peranema. Locomotion is accomplished only by creeping, the organism
being incapable of swimming. Violent metabolic movements accompany
locomotion especially when an obstacle is encountered in its path. Only
rarely is the organism stretched out straight. When the direction of
locomotion is changed or when a strong stimulus is received from another
organism the shape assumed during locomotion is entirely lost in a very
violent twisting and kneading movement, which soon, however, gives way
again to the cylindrical shape characteristic of locomotion. A very slight
stimulus is sufficient to cause very marked metabolic movements.

The exact manner in which locomotion is brought about could not be:
ascertained satisfactorily, but it was observed that the tip of the flagellum
was habitually bent in the form of a small loop which was used somewhat
in the manner of a paddle so as to pull the organism along. During
locomotion, it is worthy of note, the organism rolls over frequently
though not regularly, differing in this respect from the peranemas.

Externally, Jenningsia is radially symmetrical like the euglenas.
The body is covered with a thin cuticle which is marked with spiral
striations that take their origin in the mouth. Small slender spindle-
shaped structures about one and one-half microns in length, which may
readily swing about in the water, are attached to the striations at irregular
intervals. These cuticular spindles are most numerous near the extreme
anterior end.

Internally there are found: a large central oval nucleus about 35
microns in diameter with a central denser body about 15 microns in dia-
meter; a complicated contractile vacuole system (similar to that found
in euglenas) near the anterior end, which functions several times a minute;
a complicated pharynx provided with stiff rod-like elements; and num-
erous strongly biconcave discs or rings of clear bluish green material,
ranging from one to three microns in diameter. One or more large
vacuoles are also occasionally found in the posterior half of the organism.

Of the structures just enumerated the pharynx is of especial interest
although it is very difficult to understand its detailed operation while
feeding. In the moving organism one can see two rod-like elements
lying with their anterior ends in a clear space free from protoplasm and

A NEW DIATOM-EATING FLAGELLATE 179

protected by a strong arched structure on the anterior side, the arch
representing the lips of the closed mouth. When the animal is com-
pressed under the cover glass the mouth is forced open and three large
rods are now seen which are forced partly outwards (fig. 3). In this
process some of the small rod-like elements disappear, which may indicate
that these apparent rods are only folds of cuticle occasioned by the
closure of the mouth and the retraction of the pharynx. Although I
have seen the animal devour diatoms on several occasions I have been
quite unable to determine exactly how the rods of the pharynx operate
during this process.

Another element of interest are the numerous rings or discs scattered
throughout the endoplasm (figs. 1, 4). They are of all sizes as stated
above and are of a clear but pale bluish green color. They are unaffected
by the ordinary stains or iodine but dissolve without visible change in
solutions of H2SO,. In their general appearance, number, and behavior
towards reagents they are similar to the crystals in amebas and may
possibly be formed in a similar manner. They are not active under the
polariscope. It is probable that these rings or discs are some by-product
of metabolism.

In one culture of few but large individuals, numerous spherical bodies
were observed which stained deeply with haematein. No food was
observed in any of these individuals.

The most interesting thing about this organism from a general point
of view is its mode of nutrition. Itis completely holozoic. As indicated
by its proposed specific name, its food is living diatoms of all sizes up to
100 microns in length. I have examined in all several hundred of these
organisms and nearly all of them had from one to five diatoms, mostly
of medium size, in them. No other food objects have been observed.

The feeding process is very difficult to observe in detail, owing to the
violent metabolic movements, the activity of the complicated pharynx,
and the speed with which the food is devoured. The organism moves
along with the tip of the flagellum moving about until it comes into
contact with a diatom. If hungry, the flagellum is brought into contact
with the diatom as much as possible while the animal continues with its
forward movement. When the anterior end of the organism comes
nearly into contact with the diatom, the posterior end rears up and vio-
lent metabolic movements set in. The anterior end is brought over the

180 A. A. SCHAEFFER

diatom and is seen to spread out. The basal part of the flagellum is
also seen to move about as if it had a part in the actual swallowing of the
food particle. Presently the diatom is seen inside the flagellate, which
moves away within a few seconds, the whole process of feeding taking
place within about 20 seconds. Although I saw a number of instances
of feeding and took particular pains to see whether the pharyngeal rods
were actually protruded, or were used merely in distending the mouth, I
was unable to determine their exact function. I incline to think how-
ever, that the rods were not protruded beyond the mouth opening.
On several occasions I tested their reactions to carmine particles without
obtaining positive responses, although one specimen so tested later
devoured a diatom. All the evidence therefore indicates that Jenningsia
is a predacious animal that feeds exclusively on living diatoms.

I observed but one instance of reproduction—asexual—which was
accomplished by longitudinal splitting, beginning at the anterior end and
extending backwards. One of the daughters inherits the old flagellum,
the other grows a new one during division.

Jenningsia affords a very good example of the ease with which fun-
damental instincts and habits may be changed in phylogeny, for it is an
animal descended from plants. The exclusively predacious instincts of
this animal contrast strongly with the true holophytic mode of nutrition
of some of the closely related family of euglenas. In so far as the actual
process of feeding is concerned we have exhibited within the span of the
single order Euglenineae all the general methods of nutrition known
among nucleated organisms: true holophytic nutrition by means of
chlorophyll among euglenas; saprophytic, the ingestion of decomposing
nitrogenous foods either liquid (astasias) or solid (peranemas); holozoic,
ingestion of carbohydrate or protein materials—pieces of animal or
vegetal tissues (peranema); holozoic, ingestion of small masses of bacteria
or pieces of animal and vegetal tissues and on living protophyta (dinema);
holozoic, predacious, injestion only of living, moving organisms that,
in a real sense, have to be captured (Jenningsia). No better illustration
than this could be found of the combined plant and animal characters
of the Flagellata.

The change from a holophytic to a holozoic type of nutrition in the
Euglenineae has been made possible by the development of the pharynx.
In the euglenas the pharynx is a simple tube-like depression at the
anterior end whose chief function seems to be that of an efferent drainage

A NEW DIATOM-EATING FLAGELLATE 181

canal for the contractile vacuole. But it also serves as a means of taking
into the body small solid particles as has been shown by placing carmine
in the culture fluid. The taking in of solid matter is however not an
essential function for the euglena, but it is of interest in that it fore-
shadows saprophytic and holozoic instincts and modes of nutrition in the
so-called colorless euglenas—the astasias—and in the peranemas.

The astasias have gone a step further than the euglenas. They have
developed a larger pharynx and, we may suppose, one better adapted to
taking in liquidfood. Atthe same time they have also lost the chloro-
phyll from their bodies.

The peranemas have likewise gone a step further than the astasias in
the development of the pharynx. In these organisms the pharynx is
provided with special rods which make it possible to open and close the
pharynx, and also to act somewhat like a suction apparatus by means of
which solid matter may be eaten with despatch. Most of these organ-
isms are small, so that they are restricted in their food to bacteria or small
pieces of disintegrating organisms. In Jenningsia we have however as
a most important development, a large size, so that it is possible for it
to feed on such large actively moving organisms as diatoms in a truly
predatory manner.

_ In company therefore with a progressively developing tendency in
these organisms from holophytic to holozoic nutrition in a physiological
sense, we have also, on the morphological side, a progressive develop-
ment of the pharynx and of the size of the organism which makes possible
the capturing and eating of relatively large masses of solid food and
actively moving organisms.

University of Tenn.,
Knoxville, Tenn.

182 A. A, SCHAEFFER

EXPLANATION OF PLATE

Fig. 1. Jenningsia diatomophaga in locomotion. Body length, 180 microns,
flagellum length, 150 microns, cv, contractile vacuole; d, ingested diatoms; e, excretion
bodies; m; mouth; n, nucleus; p, pharynx; r, pharyngeal rods; v, vacuole.

Fig. 2. Photomicrograph of living J. diatomophaga in locomotion. The dark
masses represent ingested diatoms. The basal part of the flagellum is seen at the
anterior end.

Fig. 3. Sketch of anterior end compressed under cover glass to force open the
mouth, m. s, cuticular striations originating in the mouth and running spirally around
the organism; r, the pharyngeal rods.

Fig. 4. Enlarged view of “excretion” discs. a, top view; b, cross section.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL
SOCIETY VOL. XXXVII

Fic. 4

PLATE XIII SCHAEFFER

STUDIES ON AMERICAN STEPHANOPHIALINAE*
With Two Plates

ERNEST CARROLL FAUST

Since the time of O. F. Miiller helminthologists have recognized
certain distomes which are characterized by a coronet of papillae around
the oral opening. These were first classed as Armata, which group in-
cluded also all echinostome species. The more modern studies of Looss
and Odhner have shown that certain of these species are related to the
Allocreadiidae. With Bunodera luciopercae (nodulosa) as the type Looss
(1902:453) proposed the subfamily Bunoderinae for the then known
forms, Crepidostomum metoecus, C. farionis (laureatum), Distomum petalo-
sum and Bunodera luciopercae. However, the enormous extent of the
uterus crowding the entire posterior part of the body in the case of
Bunodera and the scanty coiling of the uterus entirely anterior to the
anterior testis in the case of the other species constitutes so marked
a difference that the latter species were necessarily removed from the
Bunoderinae. Odhner (1905, 1910) placed them in the Allocreadiinae.
Nicoll (1909) showed that they were more closely related to the Allo-
creadiinae than to the Bunoderinae, but that certain differences possessed
by the entire group justified the creation of a new subfamily, Stephano-
phialinae. The writer’s study of the American representatives of this
group stands in support of Nicoll’s thesis, and contributes certain data
which mark out this subfamily more definitely.

The following description supplements Nicoll’s original diagnosis.
The integument is usually aspinose, but in the case of a few representa-
tives of Stephaniphiala farionis (fig. 3) spines are found around the oral
sucker. The circumoral papillae are always six in number, altho there
is a tendency for the dorsal papillae of Crepidostomum spp. to bifurcate.
The yolk glands in this subfamily extend from the region of the pharynx
to the posterior end of the body. Moreover, the excretory bladder
extends to the anterior border of the anterior testis, whereas in the Buno-
derinae it is a small pouch posterior to the testes. In the Allocreadiinae
as at present constituted there is no common type of excretory bladder.
In the genus Allocreadium it terminates behind the anterior testis.

*Contributions from the Zoological Laboratory of the University of Illinois
No. 115.

184 E. C. FAUST

There are recognizable in the subfamily Stephanophialinae three
genera, Stephanophiala, Crepidostomum and Acrolichanus. Stephano-
phiala contains the cosmopolitan species, S. farionis (lawreatum), and
a new species, S. vitelloba. Crepidostomum contains the type, C. metoe-
cus, C. cornutum and a new species C. illinoiense. Acrolichanus is mono-
typic, being represented by A. petalosa. The type species of the Buno-
derinae, B. luciopercae, has been found in the North American perch,
Perca flavescens, by Stafford (1904:489) and also by Lander.

The writer is indebted to Professor Henry B. Ward for valuable data
and material, and to Professor Edwin Linton, Professor Henry L. Osborn,
Dr. Arthur R. Cooper and the Bureau of Animal Industry of Washington
for the loan of material.

Stephano phiala Nicoll 1909

This genus was created by Nicoll in 1909 to separate Crepidostomum
farionis of Miller (laureatum Zeder) from C. metoecus. The separation
was made on the basis of 1) disposition and number of the oral papillae,
2) position of the genital pore, 3) size of the cirrus pouch, and 4) extent
of the uterus with size and number of the ova. These are the characters
which require that Crepidostomum farionis be placed in a separate genus
from C. metoecus, but Nicoll’s definition of Stephanophiala is obviously
obscure and demands more precision on the one hand and more flexibility
on the other.

Redesignation of Stephanophiala. Minute to medium-sized Allo-
cread species with six subequal rounded papillae encircling the oral sucker
laterally and dorsally. Prepharynx and esophagus short as in the entire
subfamily; pharynx about one-half size of the oral sucker. Forking of
the gut a little distance in front of acetabulum. Testes median tandem,
larger or smaller than acetabulum. Ovary lateral or median. Uterus
composed of a few coils between anterior testis and acetabulum, with
the vagina leading directly to the genital aperture. Cirrus pouch a mus-
cular S-shaped sac which may lie entirely dorsal to the acetabulum or
may extend a short distance behind, but never extending as far caudad
as in Crepidostomum. Genital pore always anterior to the forking of
the gut. Vitellaria marginal, somewhat dorsal, from the pharynx to.
posterior end but usually encroaching on the middle field in the region
of the testes and farther caudad. Eggs few to many, averaging about
75 in length by 45y in trans-section.

STUDIES ON AMERICAN STEPHANOPHIALINAE 185

In this genus are included Stephanophiala farionis (O.F.M.) and
new species, S. vitelloba, from the Bitter Root Valley, Montana.

Stephano phiala farionis (O. F. Miiller)

This species was first described by O. F. Miiller in 1788. It has
passed thru a vicissitude of generic names including Distoma, Distoma
(Crossodera), Crossodera, Lobostome, and Crepidostomum. It has
more frequently been known as /aureatum of Zeder than as farionis, altho
the former specific designation was not applied until some twelve years
after Miiller’s name fartonis was proposed. In the Old World the species
has been described from Central Europe, Scandinavia, and England.
In the Western Hemisphere it has been reported from Canada by Stafford
(1904) and Cooper (1915) and from Yellowstone National Park by
Linton (1893). Several Salmonidae and Percidae have been described
as host of this species.

The writer’s material for the study of this species in North America
consists of parasites of Salmo mykiss lewisii, (Gir.) collected by Linton
from Yellowstone National Park and described by him (1893) and, in
addition, material collected by Dr. B. H. Ransom, from Park Co.,
Montana, in 1904, and lent by the Bureau of Animal Industry, Washing-
ton, D.C. The study of the New World material shows it to belong to
the species S. farionis, but it shows likewise that the species is widely
variable in several characters.

Detailed descriptions of the worm have been given by Olsson (1878:
24), Blanchard (1891:481) and Nicoll (1909:425) and need not be con-
sidered here. However, a summary of the constancy and variability
of certain characters may well have a place in this paper. In length the
mature worm varies from 1.2 mm. (Ransom material) to 6 mm. (maxi-
mum record of Olsson). The ventral sucker is always larger than the
oral sucker, altho no constant ratio has been found. The fluke has a
short prepharynx, a small pharynx and an esophagus shorter than the
pharynx. The gut forks a little distance anterior to the acetabulum.
The excretory bladder extends to the anterior border of the anterior
testis. The testes are median tandem, somewhat larger than the aceta-
bulum. Anterior to them is the ovary, which may be lateral or median
in position. Most variable of all is the size and extent of the cirrus pouch.
Its posterior limit in some individuals may be near the center of the
acetabulum as Nicoll has found in his material, but in other specimens

186 E. C. FAUST

it extends behind the posterior margin of the acetabulum. In all cases,
however, its internal structure is the same, and the anterior end always
extends anterior to the genital pore. The amount of coiling of the
uterus is variable. The eggs vary in number from few to about one hun-
dred. ‘They vary in size from 62 to 82y in length and 40 to 59y in cross
section.

The papillose Allocread species have been uniformly described as
aspinose. A small per cent of specimens of the Ransom collection have
spines on the integument in the region dorsal and lateral to the oral
sucker (fig. 3). They are so definite in character as to leave no room
for uncertainty. The small number of individuals of this collection so
armed makes it inadvisable to consider them as a distinct variety.

The recognition of the American specimens, especially those of Linton
and Ransom as Stephanophiala farionis shows the wide distribution of
this species. The name S. transmarina proposed by Nicoll (1909) for
the American specimens of this species consequently becomes untenable
and is to be considered as a synonym.

Stephanophiala vitelloba nov. spec.

Stephanophiala vitelloba was taken from the gall bladder of a single
specimen of Coregonus williamsoni Gir. among several dozen examined
from the Bitter Root River, at Fort Missoula, Montana, Feb. 18, 1916.
Ten specimens were found at the head of the gall duct.

The worm is quite cylindrical, but tapers in an ovoid fashion at the
posterior end. At the anterior end there is a slight bending ventrad,
so that the oral sucker is directed anteroventrad. Topping the dorsal
portion of the oral sucker are six subequal blunt papillae, about 30 yu
wide and 20 » in depth. About one-third of the distance caudad on the
ventral side the acetabulum bulges out as a truncate cone. The fluke
is 1.11 to 1.4 mm. long by 0.277 mm. wide at the middle of the body.
The oral sucker measures 0.148 mm. in trans-section and the acetabulum,
0.176 to 0.185 mm. The integument is free from spines but has small
wartose prominences.

The oral atrium leads thru a very short prepharynx into a barrel-
shaped pharynx, 74 pw in diameter and about 100 u in length. At the
posterior end of the pharynx is a short esophagus, behind which the
digestive tube bifurcates, each cecum clasping the anterior face of the
acetabulum and then running caudad to a position near the posterior

STUDIES ON AMERICAN STEPHANOPHIALINAE 187

end of the worm. The wall of each cecum is composed of about eight
to ten cells in cross section, with rounded walls lining the lumen of the
pouch (fig. 11, ce). The cecum is oval in cross section, about 35 yw in
frontal diameter and 64 u along the sagittal plane. The lumen is scarcely
as large as the cells of the cecum wall. Each cell is filled with fine
granular material and has an oval nucleus 6 yw in section. The general
appearance of the cell is vesicular, altho no vacuoles have been found.

The excretory pore is terminal. From it, extending cephalad, near
the dorsal face is a long sacculate bladder. It is slightly pouched at the
posterior end but soon becomes distinctly tubular. It continues for-
ward as a single tube to the region of the ootype where it bifurcates,
sending forward two lateral vessels. Beyond that point the system has
not been traced. The bladder has no granular inclusions.

The male genital organs consist of two testes, efferent ducts, muscular
cirrus and seminal vesicle. The testes are rounded glands lying one
behind the other on the ventral side of the body at the beginning of the
posterior half of the body. They measure about 100 uw in diameter.
Each testis (fig. 11) contains a great number of spherical cells, arranged
almost exactly in concentric rows. The cells are about 6 yw in section.
Toward the center of each gland are found various stages of maturation
up to spermatids and spermatozoa. The maturing cells occur in large
aggregates, so that they form large clumps of densely staining bodies
in the gonocoel. From the outer margin of each testis at the anterior
end a vas deferens arises. The two efferent ducts proceed separately to
the anterior end of the ovary in which region they merge into the pos-_
terior end of the cirrus sac. The cirrus is long and muscular. It bends
on itself near the middle of the acetabulum and proceeds again forward
in the region of the left cecum, ending at the genital pore, anterior to the
forking of the ceca. The portion which constitutes the seminal vesicle
is located at the S-bend in the sac. Prostate glands occur in the anterior
half of the cirrus pouch.

The female genitalia consist of ovary, oviduct, seminal receptacle,
Laurer’s canal, vitellaria and vitelline ducts, shell gland and uterus.
The ovary lies just anterior to the anterior testis, overlapping it in part.
It is spherical, lying slightly to the side of the mid-plane (figs. 5, 12, 0)
and measures 110 to 120 win diameter. A coil of the uterus lies below it.
The immature ovarian cells are similar to those of the testes, but some-
what smaller. The short oviduct proceeds ventrad (fig. 8) where it

188 E. C. FAUST

opens into the ootype. On the left of the ootype is a pyriform recep-
taculum seminalis and branching from its neck is a tubular sac, Laurer’s
canal, directed dorsad. It ends blindly near the dorsal wall. On the
right hand side is a clump of cells, eight or ten in number, with deeply
staining cytoplasm and small spherical nuclei. These constitute the shell
glands of authors. Extending lateral right and left from the ootype are
the vitelline ducts. Their plane practically divides the animal into equal
halves. The vitellaria are the most characteristic features of the fluke.
Lateral to the ceca they extend from the extreme anterior to the extreme
posterior ends. At the posterior end they bend forward inside the ceca,
extending almost to the ootype. In section they appear as distinct
chorda, the outer series large and at times divided into branches, while
the smaller inner glands constitute a single stem. Each chordum in
section is composed of aggregates of vesicular cells of irregular polygonal
outline and large spherical nuclei which display great numbers of mitotic
figures. The entire cell is rich in chromatic materials.

From the ootype the uterus coils forward, opening to the right of the
cirrus sac. It contains from four to eight eggs. The eggs are oval, with
a lemon colored shell and operculum at one end. They measure about
77 » in length by 42 uw in trans-section. The egg is filled with granular
cytoplasm and has, in addition, from twelve to twenty-four ovate vitel-
line cells.

Crepidostomum Braun 1900

This genus was created by Braun (1900) to include the species Dis-
tomum farionis (laureatum) and D. metoecus, designating papillose Allo-
creadine species, which unlike Bunodera, have the uterus confined anter-
ior to the anterior testis. The discovery of other species related to these
shows that these Old World species are to be regarded as types of distinct
genera. The present study shows the close relationship of Crepidostomum
metoecus, C. cornutum, and C. illinoiense. On the basis of a study of
these three species a redesignation of the genus follows.

Redesignation of Crepidostomum. Minute to inframedium aspinose
Allocreadine species with six oral papillae, of which the ventral pair con-
sist of laterally extending muscular processes which taper to an acute
point. Dorsal papillae showing a tendency toward bifurcation. Short
prepharynx and esophagus; pharynx minute. Gut forking occurs a
little or a considerable distance in front of acetabulum, depending on

STUDIES ON AMERICAN STEPHANOPHIALINAE 189

expansion or contraction of prepharynx and esophagus. Excretory
bladder extending to anterior border of anterior testis. Testes median
tandem. Ovary lateral or median, just anterior to testes. Vitellaria
from pharynx to posterior end. Vitelline follicles sparsely scattered
anterior to ootype. Cirrus sac large, muscular, often convoluted, ex-
tending from ootype to front of acetabulum. Genital pore ventral or
posterior to forking of gut. Uterus consisting of several coils anterior
to anterior testis. Eggs one to several, varying in size from 55 by 40.9 p
for C. metoecus to 70 by 41 w for C. cornutum. Found in various fishes in
North America and in Vespertilio lasiopterus in Europe. Young of
American species have been found in Cambarus spp.

Crepidostomum cornutum (Osborn) 1903

This species, originally designated as Bunodera cornuta, was de-
scribed from material from Micropterus dolomieu, Ambloplites rupestris,
and Ameiurus nebulosus, taken from Chautauqua Lake, New York.
It has since been found in Canada by Stafford (1904:490) in the distomu-
lum stage in Cambarus sp. and by Cooper (1915:193) as adults in Microp-
terus dolomieu, Ambloplites rupestris, and Amieurus lacustris and in
the distomulum stage in Cambarus spp.

The material on which the original description is based (Osborn 1903)
bears evidence of being not one species but three species, namely, a
Crepidostomum species which must be regarded as the cornutum type,
Acrolichanus petalosa (Lander), and a Bunoderan species, probably
luciopercae. Specimens from Professor Osborn which the writer has
been enabled to examine consist of Crepidostomum cornutum and Acro-
lichanus petalosa. The fact that the cornutum individuals have a muscu-
lar cirrus sac and a short uterus coil never encroaching on the territory
of the testes or farther caudad precludes any possibility of regarding
them as Bunoderans. While figures 1 to 6 of Osborn’s paper are ac-
ceptable as representatives of C. cornutum, fig. 7 is distinctly a Bunoderan.
Thus it becomes necessary to redescribe C. cornutum and limit the type
more definitely.

The size of C. cornutum is variable. Its length ranges from 0.9 to
3.0 mm. and its width from 0.2 to0.9 mm. While it is slightly narrower
at the posterior end than in the region of the acetabulum, the tapering
is so gradual as to be almost inconspicuous. The oral sucker is commonly
as wide as the body or even wider, its diameter varying from 0.33 to

190 E. C. FAUST

0.46 mm. The acetabulum is considerably smaller, from 0.15 to 0.31
mm. in trans-section. It lies in the anterior quarter of the body.

Of the six papillae crowning the oral sucker the ventral pair are
lateral extentsions of the sucker itself, giving the appearance on the
whole of turned flanges (fig. 14). The four remaining papillae are equal
in size and inconspicuous in detail.

The orifice leads thru a short prepharynx into a small oval pharynx
about 50 in cross section. Behind the pharynx is a short esophagus
of equal length. The digestive tract usually bifurcates some little dis-
tance in front of the acetabulum. Long tubular ceca extend to near the
caudal extremity of the body.

The excretory system as far as it has been made out consists of a long
bladder arising from a caudal excretory pore and ending near the anterior
border of the anterior testis. The tubules have not been studied.

Turning to the genital organs, large testes, capable of considerable
elongation or widening, lie tandem in the posterior half of the body.
Osborn (1903:69-71) has described slender vasa deferentia which run
from the testes along the dorsal side of the fluke, merging into a single
duct at the base of the cirrus sac (fig. 6). From ventral aspect the ovary
is seen sometimes on the right, sometimes on the left. It is oval and
considerably larger than the receptaculum seminalis. The ootype is
located in about the center of the body. Its relation to the female
genital organs is shown in Osborn’s figure 6. The vitellaria are situated
in rather definite chorda, closely surrounding the ceca on the sides.
Anterior to the ootype they are composed of minute follicles. They
usually extend from the pharynx to the posterior end of the body. They
do not encroach on the median field as do the follicles in C. illinoiense.

The uterus in the mature worm consists of several coils anterior to
the anterior testis, with a terminal portion directed forward over the
acetabulum toward the genital pore. The genital pore is ventral or
slightly posterior to the bifurcation of the gut. Osborn (1903:72) has
found its anterior end to be muscular. The eggs of C. cornutum are
not nunerous; they range up to about twenty. They measure 65 to
71 uw in length by 414 in cross diameter. The cirrus sac is a long coiled
muscular organ arising in the region of the ootype. At times its con-
volutions separate the ovary from the receptaculum seminalis. The

STUDIES ON AMERICAN STEPHANOPHIALINAE 191

writer has found both the vesicle and the ductus ejaculatorius to be ex-
tensively muscular. As is commonly found in the Stephanophialinae
the ductus is surrounded by prostate glands.

Crepidostomum illinoiense nov. spec.

This minute fluke was taken from the intestine of the crappie, Pomo-
xis sparaides (Lac.), at Havana, Illinois, July 11, 1910, by Dr. H. J.
Van Cleave for Professor Henry B. Ward, to whom the writer is indebted
for the material. A large number of specimens were secured from the
intestine of the host.

The worm is elongate in outline, with a greatest width of 0.15 to
0.18 mm. in the region of the acetabulum, posterior to which it gradually
tapers to a distinctly conical end. The acetabulum measures from 76
to 88u in trans-section, while the oral sucker is almost twice as large.
Crowning the oral sucker is a cluster of six papillae, two ventral, two
dorsolateral and two distinctly dorsal (fig. 17). The ventral papillae
emerge from the sides of the posterior margin of the oral sucker, but are
always intimately connected with an anterior folding of the sucker. Each
papilla extends laterad about i5ythen is flexed dorsad some 5 to 7y,
terminating in a distinct point. The dorsolateral papillae are triangular
with rounded corners. They extend forward and dorsad. The dorsal
papillae lie directly above the orifice. They are separated from one
another by an inconspicuous sinus. Each dorsal papilla is bifurcate,
altho the notch is not deep. Altho there is no fundamental muscular
connection between the dorsal and the dorso-lateral papillae, folds of the
integument stretch across the intervening notch, giving the appearance
superficially of a sympapillose condition. The body is unarmed.

The mouth opens thru a short prepharynx into a spherical pharynx
of 26 diameter. Behind this organ is an esophagus of equal length.
The bifurcation of the digestive tract occurs just a little anterior to the
acetabulum. ‘The ceca are long attenuate tubes reaching to the extreme
posterior limits of the worm.

The excretory system (fig. 19) was found only in sections. The pore
is caudal in position. For a short distance forward the bladder is muscu-
lar, but as it bends dorsad its lining is entirely parenchymatous. The
tube gradually becomes more and more attenuate in the vicinity of the
testes and ends just dorsal to the anterior wall of the anterior testis.

192 E. C. FAUST

The ovary is a medium-sized reniform body lying just behind the
acetabulum. In ventral view it is covered on the right side by the cirrus
pouch and on the left by the uterus. In a median line just behind the
ovary lies the ootype, and on the right, posterior to the ootype, is a large
pyriform receptaculum seminalis. The testes lie tandem in the third
quarter of the body. The anterior one is sometimes compressed longi-
tudinally so that it reaches laterad almost to the ceca. The cirrus pouch
is an extremely long muscular organ, originating some distance posterior
to the ootype and extending forward over the acetabulum to terminate
at the genital pore, ventral to the forking of the gut. It is capable of
considerable eversion. The vitelline glands at the margins of the body
extend from pharynx to posterior end of the body. Anterior to the
acetabulum they are few and small. In the region of the testes they
encroach on the median organs and fill the entire dorsal portion of the
worm posterior to the testes. The uterus is an uncoiled tube which runs
directly to the genital pore. The eggs occur singly or at most in pairs.
The mature egg measures 63 by 40 yw. The shell of the immature egg
is colorless, while the yolk follicles are yellow; the shell of the mature
egg is a dark golden yellow, concealing the color of the yolk material.

Acrolichanus Ward 1918

The name Acrolichanus was substituted by Ward (1918:396) to re-
place Acrodactyla of Stafford 1904, preoccupied. The genus at the
present time includes a single species, first described by Looss (1902:454)
as Distomum petalosum Lander. The type material was secured from
the Lake sturgeon, Acipenser rubicundus Le S., from the vicinity of Ann
Arbor, Michigan. The species has also been found by Stafford (1904)
and Cooper (1915:194, 195) in the same host in Canadian waters. It is
highly probable that Distomum auriculatum Wedl (?) of Linton (1898:
491) and Bunodera lintoni Pratt (Linton 1901:435) are synonyms of
Acrolichanus petalosa. The type D. auriculatum Wed), described from
Europe as a parasite of Acipenser ruthendus (Wedl 1857:242, 243) is
so inadequately described that it seems unwise to give it a systematic
position.

Redesignation of Acrolichanus. Inframedium Stephaliphialine spe-
cies with six oral papillae, of which the ventral pair is draped over the
anterior end of the oral sucker. Excretory bladder dilated with con--
spicuous constriction at posterior end. ‘Testes median tandem or slightly

STUDIES ON AMERICAN STEPHANOPHIALINAE 193

oblique. Ovary close behind acetabulum; vitellaria composed of sparse-
ly scattered follicles. Cirrus sac ending in a large conspicuous sphincter.

Acrolichanus petalosa (Lander) 1902

Acrolichanus petalosa has been referred to frequently in the literature
but has never been adequately described. The writer has had access
to material and records of this species from the following sources: 1)
original drawings of Lander’s type material, 2) drawings made by Pro-
fessor Henry B. Ward of material secured from Cambarus sp., at Ann
Arbor, Mich., 1893 (Ward 1894:180), 3) material on which Cooper’s
record (1915) is based, and 4) specimens of Osborn’s material from
Ambloplites rupestris.

A. petalosa is inframedium in size, averaging from 1.5 to 2.5 mm. in
length and 0.32 to 0.54 mm. in width. The acetabulum lies at the
posterior limit of the anterior third of the body. It measures 0.16 to
0.32 mm. in cross section. The oral sucker measures from 0.27 to 0.45
mm. The body is aspinose. The disposition of the oral papillae as
seen from the front and below is strikingly similar to a notched geranium
leaf. The six lobes are practically equal; the ventrals are directed caudad
and their folds continue mesad so that they meet in an acute notch just
under the oral sucker (fig. 20). A very short prepharynx leads into
a pharynx which is at times oval but is more often campanulate.
A short esophagus bends dorsad from the pharynx (fig. 21), so
that the ceca frequently appear to airse from the base of the phar-
ynx. The ceca originate some little distance anterior to the acetabulum
and extend as large pouches to the subcaudal region of the worm. The
cells of the ceca are small, from ten to fourteen being found in a cross sec-
tion.

The excretory bladder arises posteriorly from an inconspicuous caudal
pore. For a short distance it consists of a small tube, but soon enlarges
into an irregular pouch lined with a thin layer of cells (fig. 26). The
pouch ends at the anterior margin of the anterior testis, from which region
a pair of lateral tubes can be traced forward for a short distance in pre-
served specimens. The writer is indebted to Professor Henry B. Ward
for Lander’s data regarding the details of the tubule system. From
the sides of the anterior extremity of the bladder a pair of single tubules
arises. Each tubule soon bifurcates, one branch coursing forward and
the other caudad. Capillaries ending in flame cells arise along each

194 E. C. FAUST

lateral tubule. As many as six flame cells have been found anterior
to the forking of the system, while eight have been found posterior to
this separation. The anterior limit of the system is in the region of the
pharynx. .

The ovary, a medium-sized ovoid gland, is usually located lateral to
the median line, immediately behind the acetabulum. The receptacu-
lum seminalis is very minute. The testes are large ovate organs, lying
in the third or fourth quarter of the body. The ootype is median, just
behind the ovary. Laurer’s canal which is on the left (fig. 25) meets
the duct from the receptaculum seminalis and both open into the ootype
from above (fig. 27). The sparsely scattered vitellaria extend from the
pharynx to near the posterior end of the body. Their transverse ducts
empty into the ootype from a ventrolateral direction.

The uterus emerges from the posterior margin of the ootype. After
coiling once it runs forwards to the right of the acetabulum to the genital
pore. The eggs are few to several in number, averaging up to twenty-
five in some cases. Each egg measures 70 to 72 in length by 40 to
50 w in cross section. The operculum is small but distinct (fig. 23). As
in the eggs of the other Stephanophialinae, many yolk cells are found.
The small efferent ducts from the testes converge in the plane of the
ovary. The muscular cirrus sac begins at this junction. It is daucine
in shape, except for a lateral bulge in the portion dorsal to the acetabulum.
This bulge is due to the convolution of the seminal vesicle which occupies
the middle region of the cirrus sac. The ductus ejaculatorius is con-
spicuously muscular. Both the seminal vesicle and the ductus are sup-
plied with glands. The opening of the ductus lies posterior to the forking
of the gut. Itis provided with a powerful sphincter (fig. 27).

The structure of the oral papillae is of a deep-seated character. It is
made up of an outermost basement membrane, within which are several
enveloping muscle strands, the nuclei of which can be made out plainly
(fig. 28). Within the center of the papilla is a core of longitudinal
muscle strands. Both of these series are distinct from those making up
the oral sucker. .

The main features of the worm are made out in very young distomula
(fig. 29). These include the campanulate pharynx, distinct genital
cells, representing testes, ovary and anterior sphincter of cirrus sac,
sparsely scattered vitellaria, and the constriction of the excretory bladder
at the posterior end. Eyespots are also present in young worms.

STUDIES ON AMERICAN STEPHANOPHIALINAE 195.

KEY TO THE SUB-FAMILY STEPHANOPHIALINAE

1 (4) Cirrus pouch small to inframedium, mostly lying over the acetabulum; genital
pore anterior to the forking of the gut; papillae subequal ........-.-
Stephanophiala, 2.2 ai aos sna, epee 2

2 (3) Oral sucker smaller than acetabulum; testes larger than acetabulum; eggs
few to many, varying in size from 62 to 85y by 40 to 59y; integument occa-

Slonally SpImOSe #4. cc 1.25 <i eee ante tac eae = S. farionis (O. F. M.)
3 (2) Oral sucker smaller than acetabulum; testes smaller than acetabulum; eggs
EW Ril ph Wy AD Be carla aipeg seted heme eer es 98 or 8 S. vitelloba Faust

4 (1) Cirrus pouch large, extending some distance behind the posterior limit of the

acetabulum; genital pore posterior or ventral to the forking of the gut; ven-

tral papillae large, differentiated ......----- 2s ee eee eee 5

5(10) Ventral papillae extending laterad, tapering to acute points; cirrus pouch
well developed, muscular, tapering to a small ductus ejaculatorius . .

Crepidostomum ...... 6

6 (7) Dorsal papillae entire, not tending to bifurcate; pharynx very small... .

.... C. cornutum (Osborn)

7 (6) Dorsal papillae tending to bifurcate; pharynx about one-half diameter of oral

SHIR 2569 OR het CE Ae lus ea Ore int Chee ea rae Bthe nas rome teeing” Sc 8

8 (9) Inframedium in size, testes very large......... C. metoecus Braun
9 (8) Minute, testes relatively small .............. C. illinoiense Faust
10(5) Ventral papillae extending as folds over the anterior portion of the oral cavity;
ductus ejaculatorius with a powerfully muscular end . . . Acrolichanus

Sin tle ispeciesmarcatnt ears ova ce ot aace a ete versie rr en retepses A. petalosa (Lander)

Biology of the Stephano phialinae
While no faunistic-biological reconnoissance of the Stephanophialinae
will be attempted in this paper on account of the incompleteness of
many of the records, the data are nevertheless sufficiently adequate to
indicate some of the important biological relations of the group.

With the exception of Crepidostomum metoecus Braun all of the de-
scribed species are parasites of fresh-water fishes. Stephanophiala
farionis (O.F.M.), the species most widely distributed geographically,
has also the greatest host distribution. European investigators have
reported it from Trutta fario (L.), T. trutta (L.), Epitomynis salvelinus
(L.), Thymallus thymallus (L.), and Coregonus oxyrhinchus (L.), while
American students have found it in Salvelinus fontinalis Mitch., Perca
flavescens (Mitch.), Eupomotis gibbosus (L.), Boleosoma nigrum (Raf.),
Etheostoma iowae J. and M., Stizostedion vitreum (Mitch.), and Salmo
mykiss lewisit (Gir.). In addition Stafford (1904:490) has reported this.
species from Necturus maculatus. Crepidostomum cornutum (Osborn),

196 E. C. FAUST

while limited to the Eastern part of the United States and Canada, has
been found in Micropterus dolomieu Lac., Ambloplites rupestris (Raf.),
Ameiurus lacustris (Walb.), and A. nebulosus (Le S.). Acrolichanus
petalosa (Lander) has been found in various localities in the Great Lakes
and St. Lawrence Basin, in each case from the same species of host,
Acipenser rubicundus Le S. The species described as new in this paper,
Stephanophiala vitelloba and Crepidostomum illinoiense, have each been
found in but a single host in a single locality.

The normal seat of these parasites is the anterior part of the small
intestine, altho they have been recorded from the posterior end of the
intestine, the pyloric ceca, the gall bladder, and the stomach. It is also
a matter of record that the related species Bunodera luciopercae (O.F.M.)
has wandered out of its host after the death of the latter.

Sufficient data are not at hand to determine annual cycles of infection
in the host.

The life history of no one of the species of the Stephanophialinae
has been worked out. That there is an intermediate host is a certainty,
since immature specimens of Crepidostomum cornutum and Acrolichanus
petalosa have been found in abundance in the crawfish, and cysts of
immature Stephanophiala farionis have been taken from Hexagenia sp.
But the type of cercaria is not known. However, since the cercaria of
the near relative, Allocreadium isoporum, is a rhopalocercous type and
the precocious Allocread species, Cercaria macrostoma Faust, is a cysto-
cercous type, it is highly probable that the Stephanophialine species
have cercariae that are provided with heavy tails. It is evident that
the cercariae have pigmented eyes, since representative species of all
three genera of this group show the remains of these eyes in their imma-
ture stages. Moreover, since the miracidium of Bunodera luciopercae
has pigment eye-spots, it is possible that the miracidia of the Stephano-
phialinae are also pigmented.

A tentative life history scheme for the various species of Stephano-
phialinae may be outlined as follows:

STUDIES ON AMERICAN STEPHANOPHIALINAE 197

miracidium- > sporocyst-> cercaria- > distomulum- >adult distome
or with heavy

redia tail and
eye-spots
See = veer.
in snail in insect in fresh water
or fishes (exception-
crustacean ally in other ver-
_ tebrates)
IMPORTANT REFERENCES
Blanchard, R.
1891. Notices Helminthologiques. Mem. Soc. zool., France, 4:420-489; 38 figs.
Braun, M.

1900. Trematoden der Chiroptera. Ann. d. K. K. Naturhist. Hof-mus., 15;

217-236; 1 Taf.
Cooper, A. R.

1915. Trematodes from Marine and Fresh-Water Fishes. Trans. R. Soc.

Canada, (3)9:181-205; 3 pl.
Linton, E.

1893. On Fish Entozoa from Yellowstone National Park. Rept. U. S. Com. of
Fish and Fisheries, 1889-1891 :545-564; 5 pl.

1898. Trematode Parasites of Fishes. Proc. Nat. Mus., 20:507-548; 15 pl.

1901. Parasites of Fishes of the Woods Hole Region. Bull. U. S. Fish Com.
for 1899 :405-492; 34 pl.

Looss, A.

1902. Ueber neue und bekannte Trematoden aus Seeschildkréten. Nebst
Eréterungen zur Systematik und Nomenclatur. Zool. Jahrb., Syst., 16:
411-894; 12 Taf.

Miiller, O. F.
1788. Zoologica danica. 4 vol.in 2. Havniae. 225 pp., 160 pl.
Nicoll, Wm.

1909. Studies on the Structure and Classification of Digenetic Trematodes.

Quar. Jour. Micr. Sci., n.s., 53:391-487; 2 pl.
Odhner, T.

1905. Die Trematoden des Arktischen Gebietes. Fauna Arctica, 4:291-372;
Subat.

1910. Nordostafrikanische Trematoden. Fascioliden. Results Swedish Zool.
Exp., 1901. Stockholm. 170 pp.; 6 Taf.

Olsson, P.
1876. Bidrag till Skandinaviens Helminthfauna. Stockholm. 35 pp.; 4 Taf.
Osborn, H. L.

1903. Bunodera cornuta sp. nov.: a New Parasite from the Crayfish and Cer-

tain Fishes of Lake Chautauqua, N. Y. Biol. Bull., 5:63-73; 7 figs.

198 E. C. FAUST

Stafford, J.
1904. Trematodes from Canadian Fishes. 1. Zool. Anz., 27:481-495.
Ward, H. B.
1894. On the Parasites of the Lake Fish. Proc. Am. Micr. Soc., 15:173-182, 1 pl.
1918. Parasitic Flatworms, in Ward and Whipple’s Fresh-Water Biology, 365-
453, 113 textfigs.
Wedl, C.
1857. Anatomische Beobachtungen iiber Trematoden. Sitz. K. Akad. Wiss.,
Wien. Math.-naturwiss., 26:241-278, 4 Taf. .

EXPLANATION OF FIGURES

(Code ages ree hes: cirrus pouch Sy ee ee shell gland

"Cotes rue eam cecum |B Le Nera anterior and posterior testes
56 ca Pea vitelline duct EUS Sart, ener ene uterus

CEN neers egg Veet pax eet bation vitellaria

Pe ware genital pore Vl, tna anaes vas deferens

IL CS 2. eres Laurer’s canal VS nase seminal vesicle

Oi das eae ovary Se Rien excretory bladder
Die pharynx SLE eo ootype

Bea eee receptaculum seminalis

DESCRIPTION OF FIGURES

PLATE XIV

Stephanophiala farionis. 1.—Ventral view, X 54; 2.—lateral view of oral sucker
and papillae, X 105; 3.—detail of portion of integument lateral to acetabulum, showing
spines, X 180; 4.—egg, X 180.

Stephanophiala vitelloba. 5.—Dorsal view, X 54; 6.—dorsal view of head, show-
ing papillae, X 105; 7.—egg, X 240; 8.—detail of sex organs in region of ootype, X
180; 9.—cross section of worm just anterior to acetabulum, X 180; 10.—cross section
thru ovary, X 180; 11.—cross section thru posterior testis, X 180; 12.—cross section
thru region of ootype, X 180; 13.—detail of lateral papilla, X 180.

PLATE XV

Crepidostomum cornutum. 14.—Ventral view, X 34; 15.—detail of anterior end,
X 24.

Crepidostomum illinoiensis. 16.—Ventral view, X 105; 17.—detail of anterior
end, ventral view, X 180; 18.—egg, X 330; 19.—diagram of lateral aspect, X 75.

Acrolichanus petalosa. 20.—Ventral view, X 34; 21.—median sagittal section,
X 34; 22.—detail of anterior end, X 24; 23.—egg, X 330; 24.—cross section thru region
of genital pore, X 75; 25.—cross section thru region of ootype, X 75; 26.—cross sec-
tion thru anterior testis, X 75; 27.—detail of genital organs, X 50; 28.—section thru
lateral papilla, X 105; 29.—ventral view of young fluke, X 34.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL
SOCIETY VOL. XXXVII

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TRANSACTIONS OF THE AMERICAN MICROSCOPICAL
SOCIETY VOL. XXXVII

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DEPARTMENT OF NOTES AND REVIEWS

It is the purpose, in this department, to present from time to time brief original
notes, both of methods of work and of results, by members of the Society. All mem-
bers are invited to submit such items. In addition to these there will be given a few
brief abstracts of recent work of more general interest to students and teachers. There
will be no attempt to make these abstracts exhaustive. They will illustrate progress
without attempting to define it, and will thus give to the teacher current illustrations,
and to the isolated student suggestions of suitable fields of investigation.—[Editor.]

AQUATIC MICROSCOPY

To those of us who remember the first thrills of work with the micro-
scope it is not easy to understand why there are not more amateur stu-
dents of nature who cultivate a life interest in the microscope. Very
much of the early enthusiasm of this Society was given to it by those
who were using the microscope in amateur exploration of the marvels of
nature. The American schools and colleges make much use of the
microscope, but those who are most interested in these things tend to
go on into the study of some more or less technical aspect of the subject.
It would seem however, if our teaching were as effective and inspiring
as it should be, that more of our students would thruout life continue
an amateur interest in this charming field.

Perhaps, as we grow older in America and achieve more leisure and
more general interest in culture, we may again increase the number of
those who untechnically use the microscope for the mere love of its
interesting revelations. In England there are many such, and numerous
simple handbooks have been issued which helpfully guide the beginner.
Dr. Stokes has been a long-time exponent and writer for such students
in this country. His little book, now in its fourth edition, has done its
part to keep alive amateur interest. The author undertakes in the
simplest and most concrete way possible to answer the questions of the
embryo naturalist, such as:—‘How can I best find and collect the
microscopic plants and animals? How can I tell them apart and deter-
mine what they are and how they are named? What outfit will I need,
and how can I prepare my material for profitable study?”

These questions are answered in the following chapters: I, The
Microscope and its Parts; II, Common Aquatic Plants Useful to the

200 AMERICAN MICROSCOPICAL SOCIETY

Microscopist; III, Desmids, Diatoms, and Fresh Water Algz; IV, Rhizo-
pods; V, Infusoria; VI, Hydra; VII, Aquatic Worms (and Insect Larve);
VIII, Rotifera; IX, Freshwater Polyzoa; X, Entomostraca and Phyllo-
poda; XI, Water Mites and Water Bear.

The especial virtues of the book are in its untechnical language, its
simple descriptions, its concrete keys and synopses, and its outline draw-
ings. The author states that, with the exception of a few western forms,
every type of organism mentioned in the book was taken by him from a
single pond in central New Jersey. Aside therefore from its value as a
guide for the study of nature, it has a value as a contribution to intensive
study of a limited locality. The same features which make it useful to
the independent beginner will make the book helpful to the students of
elementary Biology in high school and college, and stimulate to more
effective field work.

Aquatic Microscopy FOR BEGINNERS, by Alfred G. Stokes. Fourth edition, revised and enlarged;
324 pages, illustrated. John Wiley & Sons, New York and London. Price $2.25 net, postpaid.

AMERICAN MICROSCOPICAL SOCIETY 201

Albert McCalla, M.A., Ph.D., F.R.M.S., died suddenly of heart
failure at 9:15 p.m., on Thursday, June 6th, 1918, at his late residence,
2316 Calumet Avenue, Chicago. He was 72 years of age and his demise
came after more than a year of ill health. He was the son of Thomas
McCalla, one of the first bankers of Chicago, and Marianne Davisson,
and was the brother of the late Mary Ella McCalla. Of Scotch descent,
he was of South Carolina and Virginia lineage.

Mr. McCalla was much interested in scientific research and received
a number of degrees. He was possessed of unusual expert ability with
the microscope and was the inventor of an attachment widely used in
days gone by.

He graduated from the old Chicago High School and was the winner
of the Foster Medal; graduated from Monmouth College; was one of the
founders of Beta Theta Pi at the old Chicago University. He was a
member of the following organizations:

Fellow Royal Microscopical Society, London.

Past-President American Microscopical Society.

Past-President Llinois Microscopical Society.

Member American Association for the Advancement of Science.

Member First Presbyterian Church, Chicago.

Member Sons of the Revolution.

Member Beta Theta Pi Fraternity.

He taught at Parsons College and later at Lake Forest College.

He is survived by his wife, Eleanor Hamill McCalla, daughter of the
late Honorable and Mrs. Smith Hamill, and four children, Helen Wayne
McCalla, Thomas Clarendon McCalla, Major Lee A. McCalla, U.S. A.,
and Paul Hamill McCalla.

TRANSACTIONS

OF THE

American
Microscopical Society

ORGANIZED 1878 INCORPORATED 1891

PUBLISHED QUARTERLY

BY THE SOCIETY

EDITED BY THE SECRETARY
T. W. GALLOWAY

BELOIT, WISCONSIN

VOLUME XXXVII
NuMBER FouR

Entered as Second-class Matter August 13, 1918, at the Post-office at Menasha,
Wisconsin, under act of March 3, 1879. Acceptance for mailing at the
special rate of postage provided for in Section 1103, of the

Act of October 3, 1917, authorized Oct. 21, 1918

The Collegiate Press
GrorcE BANTA PUBLISHING COMPANY
MENASHA, WISCONSIN
1918

ee ee

TABLE OF CONTENTS
FOR VOLUME XXXVII, Number 4, 1918
A New Species of Rynchelmis in North America, with Plate XVI by F. Smith
EAT el Wig BG Ml DT (ol SEES ee Me Nee SE Re ee Done Ss ea oe ED Ek Lr

Development of the Wolffian Body in Sus Scrofa Domesticus, with Plate XVII
FOP RER by bdward! |. Angles Ay Meo TD). s..cstcscss Saco see ne

Variation in the Horizontal Distribution of Plankton in Devils Lake, North
WOR Oba ab yy irik! Gx MODENE § 2ct.05- sesh orig et eee ee ee

Notes and Reviews: Genetics in Relation to Agriculture (McGraw-Hill); Nitrate
Cellulose as a Substitute for Celloidin, by Chas. H. Miller

OFFICERS

President?) Ts) is GRTRRIN ee ae el ate ie eee ee eee ee Pittsburg, Pa.
First Vice-President? 1. Mi WRELPLEV 2 ios eee ee ee ee St. Louis, Mo.
second Vice President: ‘C..O. HMsTemEw ON i es sale ee a Los Angeles, Cal.
weer etary: Lai Wa GALLO WAN ates oles ue estes coer ere ee Beloit, Wis.
Treasurer: TH. J. VAN CLBAVE 43.20.65. 3.0)don ene ee Urbana, Ill
Custodian: MAGNUS (PFLAUM. Jo.ss.0icseot eee Se Meadville, Pa.

EX-OFFICIO MEMBERS OF THE EXECUTIVE COMMITTEE
Past Presidents Still Retaining Membership in the Society

Smon Henry GAGE, B.S., of Ithaca, N.Y., :
at Ithaca, N. Y., 1895 and 1906
A. CLIFFORD MERcER, M.D., F.R.M.S., of Syracuse, N. Y.,
at Pittsburg, Pa., 1896
A. M. Bere, M.D., of Columbus, Ohio,
at New York City, 1900
C. H. E1GENMANN, Ph.D., of Bloomington, Ind.,
at Denver, Colo., 1901
E. A. BrrcE, LL.D., of Madison, Wis.,
at Winona Lake, Ind., 1903
Hnery B. Warp, A.M., Ph.D., of Urbana, IIl.,
at Sandusky, Ohio, 1905
HERBERT OsBorNn, M.S., of Columbus, Ohio,
at Minneapolis, Minn., 1910
A. E. Hertzter, M.D., of Kansas City, Mo.,
at Washington, D. C., 1911
F. D. HEALD, Ph.D., of Pullman, Wash.
at Cleveland, Ohio, 1912
CHARLES BROOKOVER, Ph.D., of Louisville, Ky.,
at Philadelphia, Pa., 1914
Cartes A. Korom, Ph.D., of Berkeley, Calif.,
at Columbus, Ohio, 1915
M. F. Guyver, Ph.D., of Madison, Wis.,
at Pittsburg, Pa., 1917

The Society does not hold itself responsible for the opinions expressed
by members in its published Transactions unless endorsed by special vote.

TRANSACTION

OF ;
American Microscopical Society

(Published in Quarterly Instalments)

Vol. XXXVII DECEMBER, 1918 No. 4

A NEW SPECIES OF RHYNCHELMIS IN NORTH AMERICA*
F. Smit AND L. B. DIcKEY

The worms described in this paper are part of a series of Oligochaeta
obtained by Miss Bessie R. Green from the vicinity of Flathead Lake in
Montana during the summer of 1914, while at the Biological Station
maintained by the University of Montana. The Rhynchelmis speci-
mens were collected in July by A. G. Vestal and M. J. Elrod, for whom
the species is named, from a creek near the Station, and included several
mature specimens and a number of immature ones.

But two species and a variety of Rhynchelmis have previously been
known. R. limosella Hoffmeister is a common European species, and
the Asiatic species R. brachycephala and its variety bythia have been
somewhat recently made known by Michaelsen (1901 and 1905). We
now describe a distinct but somewhat closely related species from North
America.

A modification of the definition of the genus in a few characters is
necessary, and a still closer relationship between Rhynchelmis and the
North American genera Sutroa and Eclipidrilus becomes apparent.

RHYNCHELMIS HOFFMEISTER

Setae simple. Spermiducal pores paired on 10.f Oviducal pores
paired in intersegmental groove 11/12. Spermathecal pores paired on
8. Longitudinal muscle layer completely separated into eight longitu-
dinal bands. Transverse blood vessels, two pairs, in each of most
somites. Spermaries and spermiducal funnels paired in 10, or two pairs
in 9 and 10; sperm ducts, one or two pair, opening into a pair of long atria.
Ovaries paired, in 11. Spermathecae paired, in 8, without diverticula
opening into the spermathecal ducts, ampullae communicating with the
alimentary tract.

* Contributions from the Zoological Laboratory of the University of Illinois, No.
106.

} Arabic numerals are used to designate the somites, counted from the anterior end.

208 SMITH AND DICKEY

RHYNCHELMIS ELRODI SP. NOV.

Length, 47-65 mm. Somites, 133-177. Proboscis long and slender.
Setae closely paired. Clitellum on 9-17. Spermiducal, oviducal, and
spermathecal pores nearly in seta line ab. Longitudinal muscle bands
not spirally rolled at edges. Ventral vessel forked in 7, and connected
with dorsal vessel in 1. First nephridia in 13. Spermaries paired, in
10. Spermiducal] funnels, one pair; sperm ducts, one pair, imbedded in
the walls of the atria. Albumen glands lacking. Spermathecae, one
pair in 8; communicating by ducts with the alimentary tract.

From the mucky banks of a creek near the Biological Station at Flat-
head Lake in western Montana.

Holotype and paratypes in the collection of the senior author (Cat.
No. 1058).

The more important facts of structure were gained from the study of
a series of sagittal sections of the 33 anterior somites of one specimen, and
of two series of transverse sections from the anterior 18 somites of each of
two other specimens, of which one is the type.

EXTERNAL CHARACTERS

Alcoholic specimens, apparently sexually mature, are 47-65 mm. in
length, and 0.9-1.25 mm. in diameter in the region of the clitellum, where
the diameter is greatest. In the anterior half of the worm the body is
nearly circular in cross section, unlike other described species of Rhyn-
chelmis, and elsewhere.it is not decidedly quadrilateral. In one appar-
ently complete specimen, the number of somites is but 133, while in
another it is 177. The number of somites in other specimens varies be-
tween these extremes and approximates 150. The anterior part of the
prostomium is prolonged into a slender tentacle-like proboscis. The
setae are closely paired and the distances between the pairs are approxi-
mately indicated by the formula; aa*:bc:dd=3:5:5. The setae are sig-
moid, slightly more curved at the distal end, slender, and simple. The
average length is about 0.27 mm., and the diameter at the nodulus is
about 0.01 mm. The nodulus is at about one-third of the length of the
seta from the distal end.

The clitellum is developed on 9-17 and encroaches slightly on the
adjacent somites. It is most strongly thickened on 10-16, and is devel-

* Letters are used to designate the setae of either side of a somite, beginning with
a for the most ventral one and proceeding in order to d for the most dorsal one.

NEW SPECIES OF RYNCHELMIS IN NORTH AMERICA 209

oped ventrally as well as dorsally. The spermiducal pores are paired on
10, slightly anterior to 10/11, and nearly in line with the ventral setae,
The oviducal pores are small, in 11/12, and in line with the ventral setae.
The spermathecal pores are paired on 8, posterior to the ventral setae.

INTERNAL CHARACTERS

The brain lies dorsad of the mouth, in the first somite, and is similar
in form to that of R. limosella, as figured by Vejdovsky (1876). The
ventral nerve cord is closely adherent to the body wall throughout its
length. The layer of longitudinal muscle fibers is in eight distinct bands,
as in other species of the genus, but the edges of these bands are not
rolled as in R. limosella (Vejdovsky, 1884, pl. 16, figs. 1 and 2), and in R.
brachycephala and its variety, as described by Michaelsen (1905:62-63).
The alimentary tract is simple in character, like that of the other species.

The ventral vessel forks in 7 and the two anterior branches unite near
the brain with the dorsal vessel. A pair of transverse vessels in the pos-
terior part of each of somites 2-6, connect the dorsal vessel with the
branches of the ventral; and similar transverse vessels in 7-12, connect
the dorsal and ventral vessels. In one specimen there is a similar vessel
on one side of 13.. The paired posterior transverse vessels of somites
posterior to 12 are connected with the dorsal vessel only. They have a
few caecal branches and often extend only part way down the sides of the
body. There is a pair of transverse vessels in the anterior part of each
of most somites posterior to 7. The first pair are somewhat shorter and
more simple, but those of somites posterior to 8 extend to the ventral
side and have several caecal branches. In the somites that have been
examined, posterior to 12, each of these vessels is connected with the
ventro-lateral wall of the intestine by a branch which extends obliquely
dorsad and mesad from that part of the vessel lying in the ventro-lateral
part of the body cavity. Ventro-intestinal vessels connect the ventral
vessel with the ventro-median wall of the intestine (fig. 1). In somites
10 or 11 to 18 or 19 inclusive, these vessels, usually three in number, enter
peculiar glandular bodies which are closely associated with the ventro-
median wall of the intestine and correspond to the blutdriisen described
by Michaelsen (1901:178) in R. brachycephala. These blood glands

(fig. 1) are more intimately united with the wall of the intestine in R.
elrodi than are those of the other species.

210 SMITH AND DICKEY

In the specimens examined, the most anterior nephridia are in 13 or
14, and they are more or less irregularly distributed posteriorly. There
are sometimes a pair in a somite, sometimes a single one, and often none
at all. Just posterior to the septum which supports the nephridial fun-
nel, there is an enlargement similar to that found in a considerable num-
ber of other species of lumbriculids. The nephridiopores are in the line
of the ventral seta bundles and a short distance anterior to them.

There is but one pair of spermaries and they project freely into 10
from their attachment to the posterior face of 9/10. A pair of sperm
sacs extend posteriorly on either side of the alimentary tract, from their
openings in septum 10/11, at least as far as to somite 30, in some speci-
mens. ‘The spermiducal organs are similar in their main features to
those of other species of the genus; but there is no trace of more than one
pair of sperm ducts or spermiducal funnels, and those present belong to
somite 10. The funnels are on 10/11, below and laterad of the openings
of the sperm sacs, and the dorsal edges of the funnels extend into the sacs,
along their ventral wall for a short distance. In tracing each sperm duct
from the funnel towards the external pore, we havea relatively slender duct
which extends posteriorly through several somites in the cavity of the cor-
responding sperm sac, to a position at which it enters the posterior end of
a much larger and tubular atrium which extends anteriorly into 10 and
then, bending ventrally, joins the body wall, posterior to the ventral
setae, and opens to the exterior at the spermiducal pore. There is a
general correspondence between the main features of the spermiducal
organs, as outlined above, and those of the other species of the genus;
but a more detailed study yields distinct differences, as will appear later.
From the funnel the sperm duct first extends ventrad along the septum
and then anteriad to the atrium which it follows closely to the place of
their union. The duct and atrium are merely in contact in somite 10,
but in the anterior part of the sperm sac the duct becomes more strongly
flattened against the atrial wall, and about opposite 11/12, in the type
specimen, it enters the tissue of the atrial wall (fig. 2, sd) and follows it
to a point near the posterior end of the atrium, where duct and atrium
merge and their cavities become continuous. ‘This intimate relation of
duct and atrium is more like the condition found in certain species of
Eclipidrilus than it is like that of the other species of Rhynchelmis.
In the type specimen the atrium extends posteriorly to 15, and in the
other sectioned specimens not so far. Numerous small glandular masses

NEW SPECIES OF RYNCHELMIS IN NORTH AMERICA 211

or prostate glands which are much like those of other species of the genus,
are attached to the outer surface of the atrium (fig. 2, pr). The ectal
ends of the atria are apparently protrusible and may function as penial
organs. In somite 9, in other species of Rhynchelmis, there are organs,
either one or a pair, which are known by various names: albumen glands,
Kopulationsdriisen, etc. There are no recognizable traces of such
organs in R. elrodi.

There is but one pair of ovaries and these are in 11 and are attached
to the septum 10/11. The paired ovisacs extend posteriorly from 11/12
and closely invest the corresponding sperm sacs except where ova prevent.
They extend through several somites posteziad of the sperm sacs. Paired
oviducal funnels are on the anterior face of the septum 11/12, and the
very short oviducts open to the exterior in the segmental groove 11/12
in line with the ventral setae. Paired spermathecae in 8, correspond
closely with those of the other species of the genus. They open to the
exterior posteriad of the ventral seta bundles of 8; the ducts are without
diverticula; and the ampullae open through narrowed duct-like portions
into the alimentary tract. In one specimen the spermathecae have no
connection with the alimentary tract and the diameter of the lumen is
much less than normal. This is probably due to degeneration, since the
spermaries are small and apparently at a stage of inactivity and yet the
sperm sacs are well distended with sperm cells.

SYSTEMATIC RELATIONSHIPS

The new species has important characters that ally it closely with the
Eurasian species of Rhynchelmis, and others in which it is nearly related
to Sutroa (Beddard, 1892; Eisen, 1888, 1891) and Eclipidrilus. The
simple pointed setae, and much elongated atria are characters shared by
all of them. In having an intervening somite between the spermathecal
and atrial somites; in the communication between the spermathecae and
the alimentary tract; and in the lack of differentiation of each atrium into
a “sperm reservoir’ or “storage chamber” and a penial organ with
narrowed connecting duct; it resembles the species of Rhynchelmis and
Sutroa and differs from those of Eclipidrilus (Michaelsen, 1901:150;
Smith, 1900:473). It is nearer to Rhynchelmis than to Sutroa, in having
the spermathecae paired and without diverticula; but resembles the latter
rather than the previously known species of the former, in having no
atrial remnants (albumen glands) in somite 9. To the writers the rela-

EM SMITH AND DICKEY

tionships to Rhynchelmis seem more significant and they include it in
that genus. One important difference between Rhynchelmis and Sutroa
disappears when we find simple, paired spermathecae, and absence of
atrial remnants in 9, characterizing the same species. It is interesting
to note that the possibility of the existence of such a species of Rhyn-
chelmis as R. elrodi has already been forecast by Michaelsen (1908:163).

“Tch bin in meinen Betrachtungen dieser Reduktionsverhiltnisse
dann noch einen Schritt weiter gegangen. Von Rhynchelmis brachy-
cephala ausgehend, sagte ich mir, dass es kein morphologisch sehr bedeut-
samer Vorgang sei, wenn nun die rudimentiren, Samentrichterlosen
Samenleiter des vorderen Paares und die verlassenen, ihrer Hauptfunk-
tion enthobenen Atrien des vorderen Paares ganz schwinden. Es wiirde
dann ein Zustand des minnlichen Geschlechtsapparates eintreten, der
mit dem urspriinglich einfachpaarigen Apparat durchaus iiberein-
stimmte.”’

In R. limosella (fig. 3) there are two pair of spermaries and spermiducal
funnels in 9 and 10, and two pair of sperm ducts joining the paired atria
of 10. In 9 there are paired organs resembling atria but without the
atrial function since no sperm ducts are connected with them. They are
the “albumen glands” and presumably represent an additional pair of
atria which in ancestors were joined by the sperm ducts connected with
the spermiducal funnels of 9. In R. brachycephala (fig. 4) and its variety
bythia, the spermaries and spermiducal funnels of 9 have disappeared
and there is a partial disappearance of the related pair of sperm ducts,
while the atrial organs of 9 are still represented. In R. elrodi (fig. 5)
there is a complete disappearance of the reproductive organs of 9, and
we have simply the single pairs of spermaries, spermiducal funnels, sperm
ducts, and atria in 10. We also have a single pair of ovaries and of ovi-
ducts which are in 11. The location of the single pair of spermathecae
in R. elrodi, two somites anterior to the one containing the male organs,
which would otherwise seem rather peculiar, is easily understood on the
assumption that this species has been derived from ancestors similar to
R. limosella._ In accordance with the views of Michaelsen, these in turn
were presumably derived from Lamprodrilus-like ancestors in which
each pair of sperm ducts had its own pair of atria independent of others.

NEW SPECIES OF RYNCHELMIS IN NORTH AMERICA 213

LITERATURE CITED
BEDDARD, F. E.

1892. A Contribution to the Anatomy of Sutroa. Trans. Roy. Soc. Edinburgh,
37 195-202.
EISEN, GUSTAV.
1888. On the Anatomy of Sutroa rostrata, a New Annelid of the Family of Lum-
briculina. Mem. California Acad. Sci., 2:1-8.
1891. Anatomical Notes on Sutroa alpestris, a New Lumbriculide Oligochete
from Sierra Nevada, California. Zoe, 2:322-334.
MICHAELSEN, W.
1901. Oligochaeten der Zoologischen Museen zu St. Petersburg und Kiew.
Bull. Acad. Imp. Sci. St. Petersburg, (5), 15:137-215.
1905. Die Oligochaeten des Baikal-Sees. Wiss. Ergebn. Zool. Exped. Baikal-
See, unter Leit. v. A. Korotneff. 1 Lief., pp. 1-68.

1908. Pendulations-Theorie und Oligochiten, zugleich eine Erérterung der
Grundziige des Oligochiten-Systems. Mitt. Nat. Mus. Hamburg, 25:
153-175.
SmiTH, F.

1900. Notes on Species of North American Oligochaeta. IV. Bull. Ill. State
Lab. Nat. Hist., 5:459-478.
VEJDOVSKY, FRANZ.

1876. Anatomische Studien an Rhynchelmis Limosella Hoffm. (Euaxes filiros-
tris Grube). Zeit. f. wiss. Zool., 27:332-361.
1884. System und Morphologie der Oligochaeten. 166 pp., Prag.

214 SMITH AND DICKEY

EXPLANATION OF PLATE XVI

Fig. 1. Rkynchelmis elrodi. Transverse section through the posterior part of
somite 17: int, intestine; bg. blood gland; vi, ventro-intestinal vessel; vv, ventral
vessel; ss, sperm sacs.

Fig. 2. The same. Transverse section through the atrium near the place of en-
trance of the sperm duct: at, atrium; sd, sperm duct; pr, prostate glands; s, developing
sperm cells. Semi-diagrammatic.

Fig. 3. Rhynchelmis limosella. Diagram showing relations of the reproductive
organs of one side: sy, spermary; sd, sperm duct; at, atrium; al, albumen gland; oy,
ovary; od, oviduct; st, spermatheca.

Fig. 4. Rhynchelmis brachycephala. Similar diagram: adapted from figure of
Michaelsen (1901:179).

Fig. 5. Rhynchelmis elrodi. Similar diagram.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL
SOCIETY VOL. XXXVII

PLATE XVI SMITH AND DICKEY

DEVELOPMENT OF THE WOLFFIAN BODY 215

DEVELOPMENT OF THE WOLFFIAN BODY IN
SUS SCROFA DOMESTICUS

Epwarp J. ANGLE, A.M.M.D.

The results embodied in this paper are from studies undertaken some
years ago. At that time it was realized that the investigation was far
from complete and publication was delayed with the hope of further
study—a hope never realized—of the source of origin of the several por-
tions of the urinary tubules. In the light of recent research the publica-
tion of the paper at this late date is largely that the illustrations may
prove of some permanent value.

HISTORICAL

The Wolffian bodies (Corps de Wolff, Urniere, Mesonephros, Primi-
tive Kidney) were discovered by Casper Fr. Wolff in the year 1759, who
regarded them as representing the embryonic period of the true kidney
(Metanephros). They received their present name from H. Rathke in
1825; this only applied to the Wolffian bodies of birds, as Rathke termed
the same organs in mammals Okensche KGrper. In 1824 Jacobson intro-
duced the name of primordialniere and discovered that in birds these
bodies secreted uric acid, which was conducted away by the allantois.
The first mention of the mesonephros in man was made by J. Frey
Meckel (1809) in his work on comparative anatomy. Meckel describes
in fairly accurate language the mesonephros of an embryo 1 mm. long;
but evidently was in doubt as to what the organ was, as he later asks the
question—“are these structures the common source from which lungs,
liver, kidneys, adrenals and sexual organs later have their origin?”

The older writers (Wrisberg, Dzondi, Oken, Emmert and Hochstetter)
had many fanciful theories regarding the role of the Wolffian body, re-
garding it either as the beginning of the kidney or as the horn of the
utereus.

Rathke (25) led the way to a true conception by discovering the origin
of the true kidney in an embryo chick of six days and showed that the
Wolffian body was related to the kidneys as the gills are to the lungs.

The glomerulus of the mesonephros was discovered by J. Miiller (30).
The honor is due Miiller for having first accurately and correctly followed
the developmental changes of this organ in a large series of embryos.

216 ANGLE

The smallest of Miiller’s embryos hada length of 20mm. In this embryo
he describes the adrenals, which are quite large and covered by the true
kidney, the kidney and ureter, the Wolffian bodies with their conducting
sexual portions. The Wolffian body is described as a long flat organ
which is in relation on its lateral surface with the sexual duct. Miiller
emphasizes the fact of the early disappearance of the Wolffian body in
man; for in his next embryo which was 3 cm. long he found between
excretory duct and sexual gland (does not mention whether testes or
ovary) a long spur which is the remaining trace of the Wolffian body.
The chief merit of Miiller’s work consists in his having discovered the réle
which the Wolfhan body plays in the development of the sexual ducts.

Among the noted early investigators was Valentin (35) whose work
principally relates to mammals. In the Wolffian body, Valentin distin-
guished two portions; an outer half which consists only of canals and an
inner portion made up principally of coiled vessles (Malpighian bodies).
Valentin adds parenthetically that in spite of great pains it is frequently
difficult to determine the direct connection of the Wolffian tubules with
the Wolffian duct. ;

When one considers the imperfection of optical instruments in use at
the period when Miiller, Rathke and Valentin lived, one can only wonder
at the accuracy of their observations. In mammals, von Baer (37) says,
that the primordialnieren arise and disappear as in birds and that their
structure clearly points to the general characters of secretory glands.

Bischoff (42) remarks that the Wolffian bodies are only to be found in
very young human embryos and that in the second month only faint
traces of the glands are to be found. This author agrees with J. Miiller,
Rathke, Jacobson, E. von Baer that the Wolffian body is an excretory
organ. Koebelt (47) observed the atrophy of the Wolffian body in man
and higher mammals and from his work concludes that the epididymis of
the male is a homologue of the epoophoron in the female.

Waldeyer (70) devoted his attention to the early developmental
changes of the Wolffian body in the chick, mammals and man and found
the phases of development in the two latter in no way different from the
former. From the fact that the Wolffian canals in their several portions
are lined with different forms of epithelium, Waldeyer came to the con-
clusion that there were two types of canals in the gland and from this
fact differentiated them into a sexual and a urinary portion; from the
former arises the epoophoron or epididymis and from the latter the

DEVELOPMENT OF THE WOLFFIAN BODY 217

paroophoron or paradidymis. This opinion was concurred in by Rathke,
Dursy and J. Miiller.

All investigators prior to 1870 regarded the Wolffian tubules as arising
either as evaginated blind sacks from the Wolffian duct or from dif-
ferentiation of the mesoblastic tissue of the middle plate.

In the year 1874 a new theory was promulgated by the independent
investigations of three men, Semper (75), Balfour (74) and Schultz (75)
who came to the conclusion that in Selechians the segmental Wolfhan
canals are in relation with the coelom by means of nephrostomes.
According to Semper these segmental canals arise from hollow invagina-
tions of the pleuro-peritoneal epithelium. Balfour regarded the canals
as arising from solid buds from the intermediate cell mass, the buds later
acquiring a lumen.

Gétte (75), working independently of the preceding authors, found in
amphibia that the Wolffian tubules arise as hollow outgrowths from the
urogenital fold of the peritoneum. This observation was confirmed by
Spengel (76) and Fiirbringer (78). Spengel and F. Meyer, working
independently discovered in 1875 that the amphibian Wolffian body
possesses peritoneal funnels and the former regards the Wolffian body of
amphibians as possessing a segmental formation and holds that its peri-
toneal funnels deserve as in Selachians, the name of segmental nephro-
stomes. Fiirbringer (78) has shown in Petromyzon that the first anlage
of the Wolffian body originates from segmentally arranged cell cords
which arise from the peritoneal epithelium.

In 1875 KGlliker (75) investigated the origin of the Wolffian tubules
of amniota which agrees with the anamnia in its essential features. In
reptiles according to Braun (77) the Wolffian tubules arise from buds
which had previously been evaginated from the coelom epithelium; these
buds are segmental as in Selachians and solid as in mammals, and become
segmental vesicles. The connection with the peritoneum soon vanishes.
Thus we see in the seventies a strange concord of opinion; in all verte-
brates which had been investigated the theory of Semper and Balfour
regarding Selachians was confirmed.

Sedgwick (80) advanced the view later that the Wolffian tubules in
the chick do not arise as peritoneal evaginations as announced by Semper
and Balfour but arise through a differentiation of the Wolffian Mesoblast.
Soon after this Weldon (83) showed for Lacerta that the Wolffian tubules
do not originate, as held by Braun, from peritoneal evagination but

218 ANGLE

according to the theory of Sedgwick. The theory advanced by Sedgwick
for birds and Weldon for reptiles was opposed by Janosik (85) who had
investigated the subject in the chick. Mihalkovics (85) agrees with
Sedgwick and Weldon that in Sauropsida the Wolffian canals do not
arise as a growth from the coelomic epithelium but by differentiation of
the Wolffian mesoblast.

Hoffmann (89) investigated the Wolffian canals of Lacerta and found
that they arise similar to those in Selachians, with the difference that the
constriction of the nephrostome from the lateral plates occurs at an
earlier period.

According to Martin (88) in rabbits the anlage of the canals is dif-
ferentiated from the middle plate and later loses connection with the
mesoblastic somites.

Kollman (92) is authority for the statement that the middle plates in
amniota are segmental. This conclusion was arrived at by surface ob-
servation and confirmed by sections. In man the same conditions could
not be directly shown but can be assumed as the Wolffian canals are
arranged segmentally and the Wolffian vesicles show segmental charac-
ters.

In the same year Field (91) made a study of amphibians. He came
to no conclusions in Amblystoma although he considers it probable that
the tubules arise fror ‘a proliferation of the peritoneal epithelium but
not from a true invagination.

DESCRIPTION

The Wolffian body is the chief occupant of the embryonic Wolffian
ridge; in Anamnia it is the chief renal organ throughout life; in Amniota
on the contrary it disappears during embryonic life, being entirely re-
placed by the true kidney (metanephros), with the exception of a small
portion of the cephalic end which is retained and becomes a constituent
of the developing sexual gland.

In its primitive form the Wolffian body consists of a series of trans-
verse tubules emptying into the Wolffian duct. As Sempter (75) has
shown for Plagiostomes there is one tubule for each body somite.

A mesonephros in the simple form in which it is first produced devel-
opmentally is retained permanently, as Gegenbaur has shown, only in
Bdellostoma, a species of Cyclostomes. Here the organ consists accord-
ing to J. Miiller of short transverse tubules whose proximal closed ends

DEVELOPMENT OF THE WOLFFIAN BODY 219

are invaginated by glomeruli and which open after a short course into
the Wolffian duct. In all remaining vertebrates the mesonephros is
metamorphosed into a more voluminous and complicated organ and shown
manifold changes over the simple form. Here we find a distal strongly
convoluted tube opening into the Wolffian duct by means of a collecting
tube; the proximal distended portion of the canal becoming a: Bowman’s
capsule and lastly a peritoneal opening leading up to the glomerulus.
This latter however is not found in the amniota as maintained by Hert-
wig (92) who claims that it is present in the three higher classes of verte-
brates. I have searched diligently for traces of this in the chick, rabbit,
cat and pig, and have found no evidence of the presence of such a canal.
The Wolffian body develops in the intermediate cell mass which is formed
when the mesoblastic somites are constricted off from the lateral plates;
it arises through a union of the median portions of the latter and is best
known under the name of middle plate. It has been amply shown that
the coelomic epithelium of the middle plate represents without exception
the anlage of the sexual glands and I shall attempt to show that the middle
plate itself represents the anlage of the excretory apparatus and that the
latter contains no traces of coelomic epithelium; thus is shown the de-
scent of the entire anlage from mesoblast.

Preceding the appearance of the anlage of the Wolffian tubules there
appears an accumulation of mesoblastic cells on the mesial and ventral
side of the Wolffian duct. These cells assume a radial appearance and
become hollowed out to form small vesicles. These vesicles were termed
mesonephric vesicles by Remak (50) and segmental vesicles by M.
Braun (77). Braun found in lizards the number of vesicles to corre-
spond with the number of segments but in birds Mihalkovics (85) has
found the vesicles more numerous than the mesoblastic somites. In
Sus I have found from two to three vesicles for each somite and conse-
quently the term segmental vesicle of Braun is inappropriate for birds
and mammals. The Wolffian vesicles are either oval or circular in out-
line when viewed in sections and are lined with columnar epithelium.
These epithelial cells have large clear and well defined nuclei and each
cell possesses a deeply staining nucleolus. In Figures 1, 2, and 3, the
relations of the Wolffian vesicles to surrounding parts are clearly shown;
Figures 1 and 2 are transverse sections from the proximal portion of the
Wolffian body of an embryo 2.5 mm. long. The Wolffian vesicle (w. v.)
is seen in the above figures to be situated ventral and medianwards from

220 ANGLE

the Wolffian duct (w. d.) and is in close relation ventrally with the coe-
lom epithelium of the middle plate. Dorsally the vesicles are in relation
with the mesoblastic somites (m. s.) and medianwards with the aorta (a).
The Wolffian duct in these figures has not as yet acquired a lumen.
Just posterior and a trifle lateral to the Wolffian duct a small blood
vessel is visible, this is the vena cardinalis (v. c.) which is closely related
to the growth of the Wolffian body. As the Wolffian body grows and
enlarges the cardinal vein is forced to assume a position dorsal to the
Wolffian ridge. Its position is readily seen in figures 7, 21, 9 and 10.
The first two of these sections are from embryos three mm. long and the
two latter from embryos four mm. long. Its shape varies greatly as will
be seen in comparing the figures 9 and 10. In embryos a little older
(5 mm.) it will be seen in figures 12, 18 and 19 that the cardinal vein
(v. c.) is situated near the dorso-median angle of the Wolffian ridge, and
is in close relation with the Malpighian bodies (m. b.) which are fully
developed in embryos of 5 mm. length.

The Wolffian vesicles (w. v.) are shown in an oblique section in figure
3, which is from the distal end of the Wolffian body of a three mm. em-
bryo. The vesicles here are in relation laterally with the Wolffian duct
(w. d.) and medianwards with the aorta (a). The small amount of
mesoblastic tissue surrounding the vesicles is particularly noticeable.

The origin of the anlage of the Wolffian canals is a subject which has
engaged the serious attention of embryologists for the past score of years
and has given rise to a voluminous literature. Among the amniota, birds
have received the attention of a majority of investigator, reptiles still
less and mammals least of all, which is not at all commensurate with their
position and importance in the animal scale. The names of Kolliker,
Renson, Kollmannm, Egli, His, H. Meyer, Nagel and Mihalkovics are
in the foreground of investigators on the development of urogenital or-
gans of mammals.

Three views have been advanced for the origin of the Wolffian tubles:

(a) The Wolffian Tubules Arise Similarly to the Tubules of Other
Glands That is as Hollow Evaginations from the Wolffian Duct. This
theory was advanced by Remak in 1850 and was accepted by Waldeyer
(70) with the distinction that the tubule only is an outgrowth from the
duct, the Malpighian body arising from the mesoblast independently and
later joins the tubule. It is only necessary to examine sections of young

DEVELOPMENT OF THE WOLFFIAN BODY 221

embryos in which the Wolffian vesicles are yet separate from the Wolffian
duct to show the incorrectness of this view (w. v., Figures 1, 2 and 3).

(b) A More Modern Theory is that the Tubules take their Origin from
an Evagination of Cell Cords or Buds from the Coelomic Epithelium of the
Middle Plate. This was first advanced by Balfour (75) and Semper (75)
for Selachinas. Among workers on amniota the adherents of this theory
are Braun (77), Weldon (83), Kélliker (79), Kollmann (82), Siemerling
(82), Sedgwick (81) and Rensen (83). Sedgwick held this theory only
for that portion of the Wolffian body which develops anterior to the six-
teenth mesoblastic somite. In reptiles according to Braun the tubules
arise from funnel shaped invaginations of the coelom epithelium which
are then constricted off from the latter and become the segmental vesicles,
and which are present in numbers corresponding to the body segments.
These vesicles secondarily unite with the Wolffian duct. The vesicle
proper becomes the future Malpighian body while the tubule arises from
a short canal which connects the vesicle with the Wolffian duct. Weldon
holds the same theory as Braun but merely makes the statement without
any evidence.

According to Kélliker in embryo chicks of the sehond day, there are
to be seen, on the median side of the urogenital ridge club-shaped buds
of epithelial cells which are growing in towards the connective tissue of
the Wolffian blastem. Kolliker observed fine fissures in these cell cords,
which he regarded as portions of the coelomic cavity constricted off with
the cells. The connection with the coelomic cavity is lost only after
the tubules have made their union with the Wolffian duct. Kolliker
observed the same in the rabbit with the exception that no fissures were
present.

Kollmann examined embryos of mouse and rabbit and confirmed in
toto the view of Kélliker. Renson (in chick, rabbit and rat) divides the
Wolffian body into two portions, a proximal extending from the seventh
to the eleventh somite and a distal, extending from the eleventh somite
to the pelvis. In the first named region the tubules arise from isolated
buds of the pleuro-peritoneal epithelium while in the latter the tubules
are differentiated from the intermediate cell mass which had previously
arisen from an ingrowth of the pleuro-peritoneal epithelium in the form
of a longitudinal plate. The cells which become anlagen of canals are
arranged around small lacunae. The lacunae are the remains of small

222 ANGLE

fissures when the longitudinal plate was constricted off from the coelom
epithelium. The remainder of the lacunae form the cavities of the
Malpighian bodies. Renson regards the pronephros and mesonephros
as being homologous organs; a view which is untenable at the present
time.

Hertwig (92) in his text book of embryology says: ‘The collective
evidence of investigators shows that the Wolffian canals arise from the
pleuro-peritoneal epithelium of the middle plate from which solid cell
cords are formed and pass in towards the side of the Wolffian duct. In
the higher vertebrates the development of the primitive kidney is to a
certain extent abbreviated, in so far as the separate cords of cells which
arise at the constricting off of the primitive segments lie very close to-
gether and constitute an apparently undifferentiated cell mass out of
which the mesonephric tubules subsequently appear to have been dif-
ferentiated. The source of its material (mesonephros) is either directly
or indirectly the epithelium of the body cavity as it has been possible
to prove in many cases in Selachians, amphibia and amniota.”

(c) The third view is that the Wolffian tubules are derived independently
of previous existing epithelium through differentiation of the Wolffian meso-
blastic tissue. This view was first advanced by Remak (50) and accepted
by His (80), Bornhaupt (67), Egli (76), Sernoff (76), Mihalkovics (85)
and H. Meyer (90). Balfour (79), Sedgwick (80) and Fiirbringer (78)
hold this view for that portion of the Wolffian body developing distal
from the sixteenth somite. Mihalkovics (85) has made a very thorough
and exhaustive study of the development of the Wolffian body in the
lizard and chick and finds no evidence whatever to substantiate the views
of Braun, Kélliker and Rensen. Mihalkovics has shown that the tubules
of chick and lizard which correspond to the seventh to the eleventh
somite inclusive arise from the coelom epithelium and that each tubule
is connected with the coelom cavity by means of a funnel shaped nephro-
stome. At the median side of each nephrostome and projecting out from
the root of the mesentery is a free glomerulus. It is admitted by all
modern investigators that the above constitutes the head-kidney or pro-
nephros which is in no way connected or homologous with the Wolffian
body. The error of Balfour, Sedgwick and others arose no doubt from
the fact that they regarded the pronephros as the anterior portion of the
Wolffian body. I have verified the work of Mihalkovics in the chick
and find no nephrostomes or free glomeruli farther distal than the body

DEVELOPMENT OF THE WOLFFIAN BODY 223

somite. Sedgwick makes the sixteenth somite the point of differentiation.
His (80) in description of embryo “a” says that the thickness of the walls
of the Wolffian duct at an early period is double the size of that structure
later on. This fact would cause him to conclude that the tubules arise
from the duct by a fold and a consequent thinning out at this point if
the collective evidence of vertebrates did not point to their formation
from the Wolffian mesoblast. Nagel (89) in his description of two human
embryos rejects the coelom theory in toto and while admitting that his
embryos were much too old to give information on this point says if it
were not for the opinion of His (see above) he would be inclined to believe
that the tubules arise as outgrowths from the Wolffian duct. In order
to prove unequivocally that the Wolffian tubules arise from the meso-
blastic tissue of the middle plate one must have embryos of such ages
which will show the complete .cycle of changes from undifferentiated
mesoblastic tissue to fully formed Wolffian vesicles. From this point of
view I am unfortunate in the selection of my subject material as in my
youngest embryos (2.5 mm.) the segmental vesicles are already well
formed and differentiated from the surrounding tissue (see w. v. figures
1 and 2). If the vesicles arose from the coelom epithelium one would
expect to find some indication of this occurrence at the point to where
they were constricted off from the latter, opposite to the vesicles, but by
observing the vesicles (w. v.) in figures 1 and 2, it will be seen that no
fissures, thinning out of the epithelium, or depression of the latter are
to be found.

The anlage of the Wolffian canals develop in a distally extending
direction and in the embryo from which figures 1 and 2 are taken the
vesicles are well formed at the proximal end. At the distal end of this
embryo the cells of the mesoblastic tissue are just arranging themselves
around a common center and no lumen is present. Another point which
adds considerable confirmatory evidence is the fact that the immature
vesicles at the distal end are no nearer the coelom epithelium than the
more fully developed vesicles of the proximal end; which should be the
case if the vesicles arose from the coelom epithelium. In the embryo
(3 mm. long) from which figures 7 and 21 are taken one finds separating
the coelom epithelium from the underlying blastem, first a compact
layer of connective tissue (c. t.) and second an intercellular space (i. s.)
each of which amounts to more than the thickness of a tubule. With the
exception of the point at which the tubules adjoin the Wolffian duct the

224 ANGLE

coelom epithelium is separated from the underlying structures in this
embryo. As previously stated, I admit that my evidence is not complete
but all the facts which I found point strongly to the origin of the Wolffian
canals from the mesoblastic cells of the middle plate. I hope in the near
future to obtain younger embryos which will unequivocally settle this
point. While the theories regarding the origin of the anlage of the
Wolfhan canals are numerous there is a corresponding scarcity of accounts
describing the changes by which the primary vesicles are metamorphosed
into a fully developed canal, ending distally in a Malpighian body and
proximally opening into the Wolffian duct. Sedgwick (80) gives the
following account which is decidedly indefinite, ‘from the inner and
dorsal wall of the vesicle a glomerulus is ultimately developed. The
whole structure grows enormously and gives rise to the Malpighian body
and complicated coils of the later Wolffian tubule. The question as to
whether or no there are outgrowths from the Wolffian duct to meet the
independently developed Wolffian tubules is not easy to answer. I am
not now in a position to give a definite answer and will merely state that
there are appearances in my sections which incline me to the opinion
that there are outgrowths from the Wolffian duct which in the case of the
primary Wolffian tubules are solid but hollow in the case of the secondary
and tertiary tubules.”

Waldeyer (65) regarded the tubule proper as an outgrowth from the
Wolffian ducts while the Malpighian body develops separately in the
intermediate cell mass and later joins the tubule. Braun (77) holds in
reptiles that there is a short connecting canal given off from the Wolffian
duct which joins the segmental vesicle and that by the lengthening out
of this canal the tubule proper is developed; while the Malpighian body
is formed from the vesicle itself. The most painstaking and the only
complete account which I can find is by Mihalkovics (85)and he gives
in detail, illustrated by a number of figures, the various changes assumed
by the vesicle in its conversion into a Wolffian tubule. He gives an
account of this process in both the lizard and the chick and as they agree
in all essential points it will serve our purpose to relate briefly a summary
of this change occurring in the chick. The Wolffian vesicles are situated
at the median side of the Wolffian duct and their contiguous surfaces
are in close contact and at the point of union, there is a melting away of
the cells and a communication is formed connecting the lumen of both
vesicle and duct. At the same time that the above is occurring the round

DEVELOPMENT OF THE WOLFFIAN BODY 225

form of the vesicle becomes flattened by the sinking in of its dorsal wall
and asa result we see in cross section, a half moon shaped body the lateral
wall of which is joined to the median side of the Wolffian duct, and its
convex wall is ventral and at the median side of the urogenital ridge, close
to the coelom epithelium and its median point directed towards the aorta.
In the concavity of the half moon is an aggregation of connective tissue
which is the anlage of the future glomerulus. The short canal which
connects the vesicle with the Wolffian duct is the anlage from which,
when fully developed, a tortuous tubule arises; while the Malpighian
body alone arises from the half moon shaped Wolffan vesicle.

This account of Mihalkovics for the chick is entirely different from
what I have found in Sus. In the pig the Wolffian vesicle assumes an
oval form with its long diameter directed dorso-ventralwards, the walls
of the Wolffian vesicle and duct being in close contact. Shortly after
this the two are connected by a short canal, which is given off from the
dorso-median wall of the Wolffian duct and uniting at the dorso-lateral
border of the vesicle. In figure 4 the vesicle (w. v.) is seen united to the
Wolffian duct (w. d.) by a short curved canal as above described. By
comparing figures 1 and 2 with figure 4 it will be seen at this stage that
the middle plate has increased considerably in size and now projects into
the body cavity and from this period on will be designated as the Wolffian
ridge. The vesicle having become oval has receded back from the coelom
epithelium (c. e.) and its long diameter is vertical to the body axis.
Otherwise the relations of the vesicle to surrounding tissues and organs
are not changed from what was described in figures 1 and 2. A lumen
in the canal connecting vesicle and Wolffian duct is not present at this
early period (4). As to the origin of this canal whether derived from the
vesicle or from the Wolffian duct I can not positively state, but it would
seem that it is derived from the latter, from the fact that its cells like those
of the Wolffian duct have taken the stain with great avidity while the
cells lining the vesicle have pale nuclei. By comparing figures 4 and 5
it will be seen that the next stage of development is brought about by the
sinking in of the median wall of the vesicle at point ‘a’ and causes the
latter to assume somewhat of an ‘S’ shape (figure 5) whereby the anlage
of the three portions of each tubule and Malpighian body can be differ-
entiated. The proximal portion of the tubule (5) is quite narrow and it
now has a distinct lumen and curves dorsally and passes under the ventral
border of the cardinal vein (c. v.) and shortly afterwards unites with the

226 ANGLE

second portion of the tubule at point 1 (figure 1). The second portion
of the tubule extends from 1 to 2 and is spindle shaped(figure 5). This
second portion curves ventralwards with a slight lateral deviation and
then becomes constricted at point 2, then makes a sharp curve median-
wards and passes over into the third portion of the tubule. This third
portion extends from point 2 to anlage of the Malpighian body and like
the first portion is quite narrow. The third portion is directed median-
wards and is parallel with the ventral surface of the Wolffian ridge. The
anlage of the Malpighian body is the expanded distal end of the third
portion of the tubule (5) and its median surface is in close relation with
the aorta (A). In figure 6, a trifle older stage is shown and the several
portions of the tubule are more clearly defined than in figure 5. From
the preceding account it will be seen that the two distal portions of each
tubule and the Malpighian capsule are derived from the Wolffian vesicle.
By comparing figures 4, 5 and 6, it will be seen that the lumen of the two
distal portions of the tubule and the Malpighian capsule are filled with
darkly stained formative cells while in figure 6 no such cells are present
in the proximal (first) portion of tubule. This fact is additional evidence
that the first portion of the tubule arises as an outgrowth from the
Wolffian duct. In figure 8 the first portion of the tubule and the Wolffian
duct are also seen to enclose these building cells but I think it purely
accidental here and believe they have migrated from the other portions
of the tubule, after union with the Wolffian duct; for in figure 3, from a
section showing Wolffian vesicles (w.v.) and Wolffian duct, the former
are seen to enclose these formative cells while the latter has a clear lumen.

Mihalkovics (85) represents the glomerulus as developing pari passu
with the tubule. In Sus this does not seem to be the case. In an
embryo of three mm. from sections of which figures 21 and 22 are taken
the canals in the proximal three-fourths of the gland have assumed their
typical curves, but the expanded distal end of tubule which is the anlage
of the Malpighian body (a. m. b.), shows no evidence of invagination.
In figure 20, the anlage of Malpighian body shown in figure 22 is seen
more highly magnified; it is to be noticed that no evidences of invagina-
tion are to be seen. In embryos from 3 to 3.5 mm. the changes relative
to the invagination of the Malpighian capsule and the formation of the
glomerulus are first to be seen. The origin of the Malpighian tuft of
vessels (glomerulus) has, so far as I have been able to ascertain, received
very little attention from workers in this field of embryology. The only

DEVELOPMENT OF THE WOLFFIAN BODY 227

detailed account I have found is by Mihalkovics (85) who accepts the
theory advanced by Gétte (74) and Fiirbringer (78) for amphibia and
Braun (77) for reptiles. Mihalkovics found in the chick that the invagi-
nation of the Malpighian capsule went on pari passu with the develop-
ment of the tubule and that first a collection of mesoblastic cells are
noticed around the dorsal wall of the capsule and these later are invagi-
nated into the capsule and become the anlage of the glomerulus. At this
period no branches are seen approaching the Malpighian body from the
aorta. Soon after invagination has occurred, groups of darkly stained
cells are to be seen among the connective tissue of the glomerulus anlage.
According to Mihalkovics these darker stained cells are first transformed
into colorless and then colored blood corpuscles; surrounding connective
connective tissue becoming the coiled vessels. Mihalkovics quotes
Romiti and Schafer as giving this origin for the blood corpuscles and
their enclosing vessel walls, for other organs. I do not doubt the perfect
physiological propriety of this view but as a matter of fact it does not
occur here. In figure 7, from an embryo 3 mm. long, the changes pre-
paratory to formation of the glomeruli are to be seen. It will be noticed.
in this figure that the aorta (a) is relatively of large size and that opposite
the median point of the Malpighian capsule, there is an evagination of
the aorta and at this point a diverticulum is given off from the latter,
which passes outwards into the connective tissue of the Wolffian ridge
and comes in close relation with the dorsal wall of the Malpighian cap-
sule. The wall of the aorta is continuous with the wall of the diverticu-
lum and the latter is seen to be filled with numerous blood vessels enclos-
ing blood corpuscles. In some cases I find no diverticulum from the
aorta, but a number of small blood vessels instead which ramify on the
dorsal surface of the capsule; preparatory to invagination of the latter
In figure 17, from an embryo of 4 mm. in length the glomerulus is com-
mencing to invaginate while in figure 16 a fully developed Malpighian
body, from a 5 mm. embryo, is shown; the glomerulus being entirely
invaginated and surrounded by a Malpighian capsule. The cells seen in
the glomeruli of figures 16 and 17 are the nuclei of the endothelial cells
of the coil vessels, and the wavy outline of the latter is seen in figure 16.
In figure 16 in the cells lining the Malpighian capsule the transition from
cylindrical to cubical and later to connective tissue is clearly shown.
Figure 13 also represents a mature Malpighian body but owing to greater
pressure there is less space between glomerulus and capsul thane is seen

228 ANGLE

in figure 16. In figures 18 and 19, from an embryo 5 mm. long the Mal-
pighian bodies are fully developed and the large branches given off to the
glomeruli from the aorta are seen.

Each fully developed Wolffian canal consists of three typical portions,
a dorsal (first), ventral (third) and middle (second) which are connected
by two sharp curves. The dorsal portion cylindrical in form affords the
connection with the Wolffian duct and then curves dorsalwards along
the lateral edge of the Wolffian ridge and then passes medianwards along
the ventral edge of the cardinal vein and approaches close to the aorta,
on the inner side of the ridge, where it makes a sharp curve and passes
over into the spindle shaped middle portion of tubule. The middle
portion passes ventralwards and then curves under the first portion and
here makes a sharp curve and passes into the anterior portion of the
tubule which is directed medianwards and passes close to and almost
parallel with the ventral surface of the Wolffian ridge and then expands
into the capsule of the Malpighian body, at the median ventral angle
' of the ridge. The above described relations are readily seen in figures
8 and 12, the first or posterior portion of the tubule extends from the
Wolffian duct (w. d.) to point designated (1) where there is a sharp curve.
The middle or second spindle shaped portion extends from point (1) to
(2) where we find the second sharp curve. The anterior or third portion
of tubule extends from point (2) to the Malpighian capsule. In figures 9
and 10 (left section) the proximal two-thirds of first portion of tubule
(t. w.) isseen. In figure 12 a complete tubule with its Malpighian body
is shown. In figure 8 the tubule is seen arising from the ventral side of
the Wolffian duct, an occurrence which I have only found two times in
examining several thousand sections of this region. In figure 12 at
point (s) (in first portion of the tubule) there is seen a sharp secondary
curve. In figure 8 from a somewhat younger embryo this secondary
curve is present but less sharply defined. I do not find a description of
this secondary curve in the writings of any author who has investigated
the Wolffian body. While each Wolffian canal shows three typical
positions it is impossible to find any two canals which are identical
throughout their entire course. In embryos of 5 mm. from sections of
which figures 18 and 19 are taken, it is no longer possible to recognize the
entire course of a tubule. As the Wolffian body develops the tubules
lengthen out and new curves arise, giving the canals a highly tortuous
and convoluted course.

DEVELOPMENT OF THE WOLFFIAN BODY 229

‘With the formation of the primary tubules and their glomeruli the
growth of the Wolffian body is by no means complete. Two factors
contribute to the further growth of this organ; first the lengthening out
of the several portions of each tubule, the intensification of the primary
curves and by the addition of new ones; second by the formation of
secondary, tertiary and quaternary canals. I shall designate as secondary
canals all tubules developing subsequent to the primary set. As the
origin of the primary mesonephric tubules gave rise to several theories,
we have likewise a number of different views regarding the origin of
the secondary.

(a) The first view—The secondary tubules and their glomeruli arise
either by fission or buds from the primary set. Either of these processes
may have as a starting point the wall of the Malpighian capsule or the
tubule itself. Braun (77) found in reptiles and Spengel (76) in amphibia
that the primary glomeruli are first divided by fissures which continue
along the course of the tubule until the Wolffian duct is reached. In
Selachians according to the statements of Sedgwick (80) and Balfour (74)
the glomeruli is the starting point of proliferation; cell buds grow out
from the latter and towards the Wolffian tubules lying in front of them
with which their blind ends fuse. After this union has been effected
they detach their other end from the parent tissue. Renson (83) held
the same view for birds but gives no adequate proof.

In discussing the origin of the secondary canals in the human embryo
Nagel (89) says one finds numerous accumulations of epithelial cells in
the middle of the sections and which might lead one to think the further
growth of the tubules occurs through differentiation of the Wolffian
tissues. But the examination of whole series of sections shows most
clearly that these epithelial collections stand in direct relation with the
previous formed canals and that they represent the solid ends of the same.
Nowhere is there to be seen the transition of the cells of the Wolffian
tissue to the epithelial cells which would be the case of the latter arose
from the former. The solid end pieces of the canals are sharply defined
from the surrounding tissues as the canals themselves. From this an-
alysis Nagel concludes that the later development of the Wolffian canals
in man occurs through a process of buds or outgrowths of the previously
formed canals. Sedgwick (80) in describing this process in the chick
does not seem to arrive at a definite conclusion but thinks that the second-
ary arise from the dorsal walls of the primary set of tubules.

230 ANGLE

(b) Second view—The secondary canals arise like the primary from
invaginations of the coelom epithelium. Fiirbringer (78) is an advocate
of this theory and says that the secondary canals arise from the coelom
epithelium on the median side of the primary canals and passes into the
Wolffian tissue in the form of cell cords which later lose their primary
connection.

(c) Third view—The secondary canals and glomeruli arise indepen-
dently of the primary through a process of differentiation of the Wolffian
mesoblast. This view was first advanced by Bornhaupt and later con-
firmed by Balfour (79) and Mihalkovics (85). My own investigations
are in perfect accord with this later view and I will attempt to show that
in Sus the secondary canals arise independent of the coelom epithelium
and primary tubules, through a differentiation of the mesoblastic cells
of the Wolffian ridge. Mihalkovics (85) in reptiles and birds finds no
evidence that the secondary canals arise from the primary through fission
or buds. According to Sedgwick (80) the secondary canals of the chick
arise dorsal from the primary and the tertiary dorsal from the secondary;
but Mihalkovics has shown that the secondary canals may arise either
ventral, dorsal or intermediate from the primary. Investigations of
the origin of the secondary canals in Sus is difficult from the fact that the
secondary canals do not appear until the primary are quite fully formed.
In figures 8, 21 and 22 the several portions of each tubule are readily
seen, no secondary canals have as yet appeared. Like the primary, the
secondary canals develop in a proximal-distalward extending direction.
In figure 21 from the proximal region of the Wolffian body of an embryo
3 mm. long, I find the first changes which lead up to the formation of
the secondary canals; midway between the spindle shaped second portion
of the primary canal and the aorta there are to be seen several collections
of mesoblastic cells which are closely packed together. These cells
take the stain with great intensity.and contrast strongly with the sur-
rounding connective tissue. I find no branches from the aorta approach-
ing these groups of cells nor any thickening or invaginations extending
in from the overlying coelom epithelium. These cells are at quite a dis-
tance from the latter and even though epithelial cords were present it
would be difficult to conceive their passage through connective tissue,
intercellular spaces and primary tubules and finally reach the designated
point in figure 21. In figure 11 from an embryo 4 mm. long we see the
next stage in the development of a secondary canal (t. w.); here a base-

DEVELOPMENT OF THE WOLFFIAN BODY 231

ment membrane is present and the darkly stained mesoblastic cells are
assuming a radial arrangement and lumen i just appearing in the vesicle.
The shape of this secondary vesicle is oblong, its width being about one-
half of its length; while cross sections of primary vesicles are nearly
round (figures 1, 2 and 3). The next period of development is also seen
in figure 11 where the anlage of a secondary canal is just ventral to the
above described vesicle and has assumed somewhat of a ladle shaped
form. By comparing the anlage of these two tubules (figure 11) it will
be seen that the median portion of the vesicle becomes the anlage of the
Malpighian body while the lateral portion becomes the tubule proper.
This occurs in much the same way as described in the primary vesicles
(figures 4, 5 and 6), although the process of differentiation of the vesicle
into a tubule is somewhat abbreviated in the case of the secondary canals.
As to the division of a Malpighian body by fissure or buds growing out
from it—I have carefully examined the sections of a dozen embryos
ranging in size from 3 to 5 mm. and nowhere find evidence of such occur-
ences. In regard to Nagel’s view (89) that the secondary canals arise
as outgrowths from the primary I can feel sure in saying that it does not
occur. One can find numerous sections similar to figure 14 which appear
like the outgrowth of a secondary tubule from a primary, but such is
not the case for by following this outgrowth in consecutive sections it
will be found to continue into a secondary tubule and the apparent blind
sack to be caused by a sharp curve which the tubule made before joining
the collective portion of the primary canal. According to Mihalkovics
(85) secondary canals in the chick are formed either dorsal, ventral or
medianwards from the primary. By comparing figures 9, 10 and 21, it
will be seen that the first portion of the primary tubule passes very close
to the lateral surface of the Wolffian ridge and then curves backward to
the cardinal vein and lies directly in front of the ventral surface of the
latter. From this it will be seen that there is but little space for secondary
canals to develop dorsal from the primary and I have only found one in-
stance of this occurrence which is shown in figure 15. The secondary
canals do not arise ventralwards from the primary for a like want of space
(figure 9) but are found to develop medianwards from the primary
(figure 11). The secondary glomeruli are situated lateral and dorsal
from the primary; the latter occupying a position near the inner portion
of the gland just dorsal to the germinal epithelium (g. e.). The relations
of primary and secondary Malpighian bodies are shown in figure 10.

232 ANGLE

In the chick of five or six days Mihalkovics finds from 12 to 18 Wolfhan
tubules opening into the Wolffian duct in each body somite and that it
is no uncommon occurrence to find three tubules emptying into the duct
in the same section and besides the tubules which open direct into the
Wolffian duct he finds from 20 to 40 indirect tubules in each somite.
These indirect tubules empty into the collective (first) portion of a direct
canal. This would make a total of from thirty to sixty direct and indirect
tubules for each body somite. I find in Sus from 2 to 3 tubules emptying
into the Wolffian duct in each body somite. In embryos of four, five,
eight and fifteen mm. respectively the number of direct canals remains
practically the same, that is two to three to each somite. In embryos
of four to five mm. length (figures 10, 11-18, 19) from two to three
Malpighian bodies are to be seen in each section in the middle two-thirds
of the Wolffian body. In embryos ranging in size from 8 mm. to 1-5
10 cm. one frequently finds from six to eight Malpighian bodies in a single
section. From this one naturally comes to the conclusion that all or
nearly all of the secondary canals in Sus are indirect; emptying into the
collective portion of a primary canal. The examination of a number
of sections demonstrates the correctness of this as can be seen in figures
14 and 19. In figure 11 the proximal end of the anlage of a secondary
canal is in contact with the median wall of a primary tubule and later
will open into it. I have only found one instance in which two tubules
open into the Wolffian duct in the same section. This is shown in figure
15, the outer of the two tubules being a secondary while the inner is
a primary one. Thus it appears that an occasional secondary tubule
opens directly into the Wolffian duct, but is quite a rare occurrence.
Lincoln, Nebraska.

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MECKEIL, J. FR.
1809. Beitrige zur Vergleichenden Anatomie. Bd. I. 1809.

MIHALKOVICS.
1885. Untersuchunger tiber die Ent. des Harn u. Geschlechtsapparates der
Amnioten. Int. Monat. f. Anat. Bd.II. 1885.

Mrnort, C.
1892. Text-book of a human Embryology. William Wood & Co. 1892.
MEYER, FR.
1875. Beitrag zur Anatomie des Urogenitalsystems der Selach. u. Amphibien.
Sitzungsber. der Naturf. Ges. zu Leipzig. 1875.
MULteEr, J.
1830. Bildungsgeschichte der Genitalien aus Anatomischer Untersuchungen
Embryonen des Menschen u. der Thiere. Duseldorf. 1830.

NAGEL, W.
1889. Ent. des Urogenitalsystems des Menschen. A. f. M. A. Bd. XXXIV.
1889.
RATHEE, H.
1825. Beobachtungen u. Betrachtungen uber die Ent. der Geschlechtswerk-
zeuge etc. Neue Schriften d. Gesellsch. in Danzig. Bd. I.
REMAK.
1850. Untersuchungen iiber die Entwickelung der Wiebelthiere. Berlin. 1885,

RENSEN, G.
1883. Development of head Kidney & Mesonephros in Birds and Mammals.
A. f. M. A. XXII. 1883.
RUcKERT, J.

1892. Entwickelung der Excretionsorgane. Ergebnisse der Anatomie und
Entwickelungsgeschichte. Bd. I. Wiesbaden. 1892.

SEDGWICK, A.
1880. The development of the kidney in its relation to the Wolffian body in the
chick. Q. J. M.S. Vol. XX. 1880.
1881. Early development of Anterior portion of the Wolffian duct and body in
the chick. Q.J.M.S. Vol. XXI. 1881.
SEMON, R.
1891. Urogenitalsystem. Jena Zeit. Naturw. Bd. XXVI. 1891.
ScHAFER, E. G.
1890. Quains Anstomy. Tenth edition. Vol. I. Part 1. 1890.
ScHuttz, A.
1875. Zur Ent. des Selachieries. A. f. M. A. Bd. XI. 1875.

DEVELOPMENT OF THE WOLFFIAN BODY 235

SEMPER.
1875. Des Urogenitalsystem der Plagiostomen und seine Bedeutung fur das der
iibrigen Wirbelthiere. Arb. Zool.-Zoot. Inst. Wurzburg. 1875.

SIEMERLING.
1882. Beitrige zur Embryologie der Excretionsorgane des Vogels. Marburg.
1882.
SERNOFF.
1876. Beitrage zur Anatomie und Ent. der Geschlechtsorgane. Inaug. Diss.
Zurich. 1876.
SPENGEL.

1876. Des Urogenitalsystem der Amphibien. Arb. aus d. Zool.-Zoot. Inst.
Wiirzburg. Bd. III. 1876.
VALENTEN, G.
1835. Handbuch der Entwickelungsgeschichte des Menschen. U.S. W. Ber-
lin. 1835.
WALDEYER, W.
1865. Anatomische Untersuchung eines Menschlichen Embryo von 28-30 Tagen.
Leipzig. 1865.
1870. Ejierstuck und Ei. Leipzig. 1870.
WELDON.
1883. Note on the early development of Lacerta Muralis. Q. J. M.S. Vol.
XXXII. 1883.
WIEDERSHEIM, R.
1890. Urogenitalsystem, Reptiles. A. f. M. A. Bd. XXXIII. 1890.
WHE, J. W. von.
1889. Excretory organs Selachians. A. f. M. A. Bd. XXXIII. 1889.
Wotrr, C. Fr.
1759. Theoria Generationis. Halae. 1759.

ANGLE

LIST OF REFERENCE LETTERS

Ofovsadssasvoresttesseienass aorta
IND BAe es anlage Malpighian body
ELE Sere ele as ee coelom or body cavity
CON Ey, capsule
COE tee Stee coelom epithelium
CHE greet ak a notochord
GE ees eae connective tissue
Pee Oi tary ARM A epiblast
[Aer te i ta glomerulus of the Malpighian body
(dies WAS As EAE ab genital epithelium
PLR See hee BAPE Sarees genital ridge
tes ear eees intercellular space
| tine aa ve athe A! hypoblast
(| ROR eenerie eat tA Malpighian body
NILE See ee medullary canal
WIRES oir c sesso ccnascceseeate mesentery
GED ee ra or es eae middle plate
MS Ie Be cet hak mesoblastic somite
oer eRe mesoblast
NS a ene ae spinal chord
SONGS 2 Pease somatopleuric layer of mesoblast
SD MR cic a splanchnopleuric layer of mesoblast
| CRRA SE see nee ac intestine
PW sorereiese ss esas Wolffian tubule
ie Biter aaeehee primary Wolffian tubule
1a os RRO Sear A secondary Wolffian tubule
DG sree coh cst setiow: Cardinal vein
RIS Scent erence eee spermatic vein
UD Rie Rene tes Wolffian duct
RED Mae seater en ey est Wolffian vesicle

LP eee Hebe Wolffian ridge

DEVELOPMENT OF THE WOLFFIAN BODY 237

EXPLANATION OF PLATE XVII

Fig. 1. Cross section form the proximal end of the Wolffian body of an embryo
2.5mm.long. IIJ—4 x 100.

Fig. 2. Left side of figure 1 more highly magnified I—5 X 190.

Fig. 3. An oblique section passing through the distal end of a 3 mm. embryo.
The Wolffian duct and three Wolffian vesicles areshown. III—5 X 280.

Figs. 4 and 5. Cross sections from the distal end of a 4 mm. embryo. I—S5
x 190.

EXPLANATION OF PLATE XVIII

Fig. 6. Cross section from the distal end of a4mm. embryo. I—5 X 190.

Fig. 7. Cross section from the middle third of the Wolffian body of a 3 mm. em-
bryo. III—4 x 100.

Fig. 8. Cross section through the middle third of the Wolffian body of a 3 and
5-10 mm.embryo. III—3 x 140.

EXPLANATION OF PLATE XIX

Fig. 9. Cross section through the proximal end of Wolffian body of a 4 mm.
embryo. I—5 X 190.

Fig. 10. Cross section through Wolffian bodies of middle third of a 4 mm. em-
bryo 1—3 X 66.

EXPLANATION OF PLATE XX

Fig. 11. Cross section through the middle third of the Wolffian body of a4 mm.
embryo. IV—3 X 125.

Fig. 12. Cross section through the distal end of a 5 mm. embryo. IV—3 X 125.

Fig. 13. Shows the Malpighian body seen in fig. 12 more highly magnified. III—
5 X 280.

Fig. 14. Cross section through the middle third of Wolffian body of a 4 mm.
embryo, showing Wolffian duct and proximal portions of Wolffian tubules. I—S
xX 190.

EXPLANATION OF PLATE XXI

Fig. 15. Cross section through the middle third of Wolffian body of a 4 mm.
embryo, showing Wolffian duct and proximal portions of Wolffian tubules. I—S
x 190.

Fig. 16. Cross section through a fully developed Malpighian body of a 5 mm.
embryo. I—5 X 190.

Fig. 17. Cross section through a Malpighian body in which the glomerulus is un-
dergoing invagination froma4mm.embryo. I—7 X 300.

238 ANGLE

EXPLANATION OF PLATE XXII

Fig. 18. Cross section through middle third of the Wolffian body of a5 mm. em
bryo. X 160.

Fig. 19. Cross section through the distal end of a Wolffian body of a 5 mm. em-
bryo. I—4 X 39.

EXPLANATION OF PLATE XXIII

Fig. 20. Anlage of Malpighian body before invagination of capsule has occurred.
From anembryoof3mm. III—5 X 280.

Fig. 21. Cross section through the proximal end of Wolffian body of a 3 mm.
embryo. IV—3 X 125.

Fig. 22. Cross section through the middle third of the Wolffian body. From the
ame embryo as Fig. 21. III—4 X 125.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL
SOCIETY VOL. XXXVII

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HORIZONTAL DISTRIBUTION OF PLANKTON 239

VARIATION IN THE HORIZONTAL DISTRIBUTION OF PLANK-
TON IN DEVILS LAKE, NORTH DAKOTA
ERIK G. MoBerc!

The horizontal distribution of plankton has been studied by several
investigators and varied results have been obtained. It is usually held,
however, that under uniform physical conditions the distribution of
the plankton is uniform. The question is of great importance since in
quantitative plankton investigations the amount of plankton found at
a certain station is usually taken as representative of a large area.

For several years plankton studies have been carried on at the Biolo-
gical Station at Devils Lake, North Dakota, under the direction of Dr.
R. T. Young, and in connection with these studies collections were made
during the summer of 1914, to determine whether the organisms in Devils
Lake showed any diurnal movements. For this purpose collections were
made shortly after noon, just after sunset, and shortly before sunrise
from the surface, the 0.6 m., the 2.1 m., and the 3.6 m. levels, the depth
of the lake at that place being about 4m. All the samples were taken
in identically the same place and on all occasions the velocity of the wind
and the condition of the sky were similar. The samples, each of 500
cc., were concentrated to 10 cc., and counted according to the Sedgwick-
Rafter? method. In each case the total number of individuals in two
or three cells, and therefore in 100 cc. or 150 cc. of the original sample,
were counted. The results obtained are shown in Table I.

TABLE I
SHOWING THE NUMBER OF INDIVIDUALS PER LITER OF WATER

1:00-2:30 p.m. 8:30-9:30 p.m. 3:00-4:00 a.m.
Depth Crustacea | Rotifera | Crustacea | Rotifera | Crustacea | Rotifera
NUMACe ee 40 310 110 770 160 480
Oot vee Ls 20 440 90 * 920 240 560
7 Hie Wh ri Veen Bee. 25 380 95 680 280 360
3:6) mireo eee 125 330 270 940 260 580
Total all levels.. 210 1460 565 3310 940 1980

+ Numerals refer to notes beginning on p. 265.

240 MOBERG

Even if there had been a vertical movement of the organisms the total
number of individuals of one series should be approximately equal to that
of another series. Instead we find that the evening series contains more
than twice as many crustaceans as the noon series, and that collected in
the morning more than four times as many. In the case of the rotifers
the variations are not quite as large. These results seem to show that
there had been horizontal movements of the plankton animals during the
intervals between the periods of collecting.

To test the horizontal distribution further and more directly several
series of collections were made during the summers of 1914, 1915 and 1916.
In some cases the samples were taken from a number of nearby points
in a part of the lake where the physical conditions do not vary appreci-
ably, while in others the entire series was collected from a fixed point at
short intervals of time. The accompanying maps show the main part
of the lake and the approximate locations of the stations at which the
samples were obtained.

In connection with some of the 1915 series the amount of phyto-
plankton and of dissolved chemicals (oxygen, free and albuminoid am-
monias, and CO* and HCO® ions) were determmed? in order to show the
relation between the zooplankton and the food and chemical consti-
tuents in the water.

During 1914 and 1915 the Sedgwick—Rafter method of concentrating
and counting was used in the plankton work, ten squares being counted
in the case of the plants, but in the case of the animals the entire number
of individuals in five cells (one-half of the collection) was counted.
Except where specified the volume of each sample was 500 cc. and in all
cases it was concentrated to 10 cc. During 1916 the collections were
made by means of a pump anda plankton-net. The water was measured
by means of a water meter and pumped thru the net where the organisms
were retained. Five gallons (18927 cc.) were collected and concentrated
to 18.9 cc., making a ratio of 1000 to 1. In five cells of each sample the
entire number of zooplanktonts was counted.

The results of the measurements and of the analyses are expressed as
follows: depths in meters; temperatures in degrees Centigrade; oxygen
in cubic centimeters per liter; ammonias as parts per million of nitrogen;
carbonates as parts per million of CO? or HCO? ions; Nodularia (the only
filamentous alga) in number of millimeters per liter; other algae (includ-
ing Coelospherium, Gomphospheria, Dictyospherium, Chroococcus,

HORIZONTAL DISTRIBUTION OF PLANKTON 241

Merismopedia, and a number of others, less common), in standard
units! per liter; diatoms, rotifers, and crustaceans in number of indivi-
duals per liter.

The variation in the horizontal distribution of the plankton may be
studied from three different points of view; namely: (1) the variation
of the total amount of plankton, (2) the variation of each species, and
(3) the correlation between the zooplanktonts, the phytoplanktonts,
and the physical and chemical conditions of the environment.

ES ST Pe S- sx, te
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a =
| MAIN PORTION OF So. 4
| DEVILS LAKE,MD. Bx o
| Lxplanarions: ; =
— Shore Lyne 17 1883 <
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DEVILS LAKE INDIAN SPISY

FIGURE 1

242 MOBERG

The Sedgwick—Rafter method hardly lends itself to the study of the
total amount of plankton but it may be roughly estimated by considering
the occurrence of the more important species. The number of individuals
of the different species, however, may be determined quite closely.

° Biologica l Station

MAP
Showin 2g the Portion
of Devils Lake tn
wArtch the Plankion
Distribution was
Studied,

Explanations:
A-, B-B, ete. = No. of Series
2,3, etc. = » *# Sample.

0 100 200 sha aie el

Scale of Melers.

&

FIGURE 2

HORIZONTAL DISTRIBUTION OF PLANKTON 243

Whipple’ states that the experimental error is not more than about ten
per cent. In the case of some of the algae, the error is probably larger,
especially since only ten squares were counted, and in considering the
results this should be remembered; but for the animals it is probably less
since a large portion of the sample was examined. Since in most cases
the depth and the temperature were measured and the amount of chemi-
cals determined, the results give some conception of the relation between
the plankton and the environment. It must be remembered, however,
that some of the variation in the chemicals is due to experimental error.

NOTES ON THE DIFFERENT COLLECTIONS AND TABULATION OF RESULTS

Series A. A set of eight samples was collected on August 19, 1914
from points lying in a straight line between the two shores of Creel Bay
The distance between each two points was about 100 meters and the
time required for the whole series was about a half hour. The greatest
depth between the two shores was 4.5 meters at points 5 and 6.
At no point does the depth vary more than one meter and the character
of the bottom is uniform, the points 1 and 8 being outside the littoral
zone. At the time of collecting the sky was clear and there was almost
no wind. Only the animals were counted and the results are shown in
Table IT.

TABLE II
SHOWING DATA FoR SERIES A

(Amt.=Amount. % var.=variation from the mean in per cent)

Sample 1 Sample 2 Sample 3 Sample 4

)|)s | |S | |§ | — | ucx-_

Memperature<:-. sch 21.8; +3.3} 21.5} +1.9) 21.0] —0.4; 21.0} —04

Brachionus satanicu......... 108 | —39.7| 48 | —73.2| 128 | —28.4| 140 | —21.8
Brachionus miilleri.......... 44 | +62.9| 40 | +48.1) 36 | +33.3} 24 | —11.1
Pedaliont/2ei.c a anne, 248 | —32.6| 540 | +46.7| 300 | —18.5) 324 | —11.9
Moirias.2 2420 te aes rane 104 | —13.3} 40 | —66.6| 16 | —86.6} 92 | —23.3
Cyclops: 25. eee ee 16 | +10.3) 4 | —69.2} 4 | —69.2) 24 | +846

Diaptomwus...........00-....| 0 |—100.0) 4 |+100.0|/ 8 |+300.0} 0 |—100.0
1a) LT eas iene ke le 80 | +66.6| 36 | —25.0| 28 | —41.7| 48 0.0

244 MOBERG

TABLE II (Continued)

Sample 5 Sample 6 Sample 7 Sample 8

SSS SSS, Ess eee ee

Temperature..........| 20.8) —1.4| 20.8] —1.4| 21.0] —04) 21.8) +3.3] 21.1

Brachionus satani-

CUS.crcccseeeeeeeee.-.-| 356 | +98.9| 292 | +63.1} 200 | +11.7| 160 | —10.5} 179.0
Brachionus miilleri} 16 | —40.7/ 36 | +33.3} 4 | —85.1) 16 | —40.7| 27.0
iPedalion ese... 564 | +53.2| 372 +1.1) 372 +1.1} 224 | —39.1} 368.0
IMOITIA 2 Oates 44 | —63.3} 148 | +23.3| 144 | +20.0} 372 |+210.0] 120.0
KY COPS 5.22023 Sssdeses 24 | +84.6} 28 | +93.1 4 | —69.2} 12 —7.6| 14.5
Diaptomus}........... 4 |+100.0/; 0O |—100.0) 0O |—100.0) 0 |—100.0 2.0
Nauplii....................) 84 | +75.0} 48 0.0; 16 | —66.6| 44 —8.3| 48.0

These analyses show a large variation of all the species and especially
of the Crustacea. The total number of animals is almost constant, how-
ever, since one form is numerous where another is scarce. ‘The tempera-
ture varies one degree but does not seem to have any effect on the num-
ber of animals. The variations shown by the different species are sum-
marized in Table III.

TABLE III
PERCENT OF VARIATION FROM MEAN OF SERIES A

Brachionus satanicusS.............0.-c.c+-c-+ +43.4 +98.9 —73.2 124

Brachionus miilleri.........0...0....ceeee + 44.4 +62.9 —85.1 148.0
Ped alionumes meetin te sercleees eo Re ae SS +53.2 —39.1 92.3
INEQIN OMe eee ce rte Tee Reh + 63.3 +210.0 —86.6 296.6
Cyclops Meco cs tes mesos tei sess eenke +61.0 +93.1 —69.2 162.3
ING Uplii ees ey ee otra ras oe +35.4 +75.0 — 66.6 141.6

Series B. These samples were collected on August 25, 1914, in the
same locality and under the same weather conditions as those of series A,
but the distances between the different points of series B were about
twice as large, and 1000 cc., instead of 500 cc., were concentrated. No
separate counts were made of the different species but the animals are
grouped under Crustacea and Rotifera. The Crustacea include: Moina,

HORIZONTAL DISTRIBUTION OF PLANKTON 245

Diaptomus, Cyclops, and Copepod Nauplii. The Rotifera include:
Brachionus satanicus, B. miilleri,’ Pedalion, and a few Asplanchna. The
results are shown in Table IV.

TABLE IV
SHOWING DATA FOR SERIES B

(Amt.=amount. % var.=variation from mean in per cent)

Sample 1 Sample 2 Sample 3 Sample 4

De Oe nl

Amt. |% var| Amt.| % var.| Amt. |% var.| Amt. |% var. | Mean

Temperature.......... 1G etd becereto ea fe al ebye] bea eerste Mi WS eto| Leer eee 15:2 [tae
Crustacea.............. 198 |—44.7| 355 —0.8) 552 | +54.2] 326 —8.9| 358
Rotitera...........0-- 162 |—46.2| 427 | +41.8} 316 +4.9! 298 —1.0| 301

For the Rotifera the mean variation from the average is +23.4%
the maximum variation +42%, and the minimum variation —45.7%,
making a range of 87.7%. For the Crustacea the figures are: mean
+27.1%, maximum +54.3%, minimum —44.6%, range 98.9%. Here
again the crustaceans show a larger variation than the rotifers, altho the
former are more numerous.

Series C. This series was collected on June 6, 1915, from points
lying in a straight line parallel to the shores of Creel Bay, point 1 lying
just south of where the collections of series A and B were made, and point
5 a short distance inside the mouth of the bay. The sky was clear, the
time required about one hour, and the distance between each two points
about 250 meters. The results are shown in Table V.

The very slight variation in depth shows no effect upon the organisms.
The chemicals, excepting the ammonias, show a uniform distribution, the
variations not being greater than the errors of sampling and analysing.
None of the plankton forms, nor the plankton as a whole, show any rela-
tion to the amount of ammonias. It may be noted that most of the
forms were scarce at point 1, and abundant at point 5, but at the inter-
vening points the total amount of plankton appears quite constant.
Table VI summarizes the variation of the different plankton forms.

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HORIZONTAL DISTRIBUTION OF PLANKTON 247

TABLE VI
PRECENT OF VARIATION FROM MEAN OF SERIES C

Mean Maximum | Minimum Range

TES LE a: eae a ane eS ED ah SID +93.4 —45.7 139.1

Other Algae: Maas We eae ore +55.9 —49.2 105.1
Brachionus satanicus................:.00 s20v 8 +56.2 —53.1 109.3
Brachionus miilleri..................00:006 +40.0 +66.6 — 66.6 133.2
Redaliont): icin sesan ny eas +29.9 +50.5 —41.1 91.8
Aaplanehaa ied: eee 2 ies ies +118.7 — 100.0 218.7
Gyclops25/5 Ge eee een ee +48.0 +80.0 —60.0 140.0
Dipptomasit)...5 sokes ee. cesccsentatincrs + 26.6 +66.6 —33.3 99.9
Nailin pets eee tate sere enees + 33.0 +44.9 —45.6 90.5

Series D. This series was collected on June 21, 1915, well out in the
main part of the lake as shown on the map. A very slight south-west
wind was blowing and the sky was clear. The time required was about
one hour and the distance between two points about 200 meters. Table
VII shows the results.

These analyses show a uniformity of physical and chemical conditions,
except in the case of the free ammonia which varies to an unusual extent.
Since it is present in small amounts it is probable that the greater part of
its variation is due to experimental error. No relation is shown between
the amount of ammonia and the amount of plankton. All the animals,
and especially the adult crustaceans, occur in small numbers, so that
some of them will be excluded in tabulating the variation percentages.
It is important to note that at point 3 all animals, except Cyclops and the
nauplii, are absent, while at point 4 most of them are quite numerous.
The summary of the variations is shown in Table VIII.

TABLE VIII
PERCENT OF VARIATION FROM THE MEAN OF SERIES D

Mean Maximum | Minimum Range

INodnlariany scmete end oe cl +51.9 +61.2 —72.1 133.3
Other Alode hana tek so. =paleles +22.3 —14.6 36.9
Brachionus satanicus..............-ccc-000-- +138.1 +276.2 — 100.0 376.2
Brachionus miilleri......0.0... cece. +81.8 +118.2 —100.0 218.2
Redalions): Ae War core Uaen fae nue ee ae 5/( +77.1 — 100.0 Viel

Nauplii.. 3.2 iie eee ear eee +50.0 +100.0 —80.0 180.0

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HORIZONTAL DISTRIBUTION OF PLANKTON 249

Series E. This series was collected on July 27, 1915, from an an-
chored raft some distance from the shore, where the depth was about
four meters. The four samples were taken at fifteen minute intervals
beginning at 11:30 a.m. and continuing until 12:15 p.m. The wind was
blowing from the south causing small waves, which increased in size
toward the end of the series. Cloudiness and sunshine alternated at
short intervals thruout the period. The results of the analyses are
shown in Table IX.

The temperature and the chemicals are almost constant. The first
sample shows a small amount of both plants and animals while in sample
4 the zooplanktonts are abundant. Table X summarizes the percentages.

TABLE X
PERCENT OF VARIATION FROM MEAN OF SERIES E

Mean Maximum | Minimum Range
Moudilariae stot 180k nae e kee +10.8 +10.5 —21.5 32.0
AEH OES ACh ns Sertteceec seat sas tees eae ae 740 +14.0 —9.0 23.0
GHAELOCEIOS Seon eee + 34.7 +36.1 —42.9 79.0
@eEnem GIAtOMS oF oo ae 3 scteasseess, +69.5 +139.0 —65.9 204.9
Brachionus satanicuS..........ccccccecceee- + 39.3 +78.6 —41.2 119.8
Brichionussmullent... 79.8 oe: +28.2 +54.3 —54.3 108.6
Bedalioui. eee eee eh +17.8 +35.6 —16.3 51.9
Gyclopsh sec ee ae UE ee! +25.0 +50.0 —50.0 100.0
PAPLOMLOS Ste eee ee ae ae! +50.0 +75.0 —100.0 175.0
Naa tieecs ee eee cee oe IED, +73.1 —70.1 143.2

Series F and G. These two series were collected on August 3, 1915,
at the same point as was series E. Series F represents samples taken
from the surface, while the samples of series G were taken from a depth
of three meters. The samples of the two series were taken alternately
at fifteen minute intervals, the period between the collecting of two sam-
ples of the same series therefore being a half hour. The first collection
was made at 2:00 p.m. The sky was clear and there was almost no wind.
The results of the analyses of series F are shown in Table XI.

These analyses show the physical and chemical factors to be quite
constant, and the total amount of plankton seems fairly evenly distri-
buted, except in 1 where all the animals and most of the plants are absent
or few in number. The results are summarized in Table XII.

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252 MOBERG

TABLE XII
PERCENT OF VARIATION FROM MEAN OF SERIES F

Mean Maximum | Minimum | Range
Nodularia la on wetats, shes + 22.2 +444 —21.9 66.3
OthertAlyaeres ne ey oe ee ee + 20.3 +27.2 —37.1 64.3
GHAGEOCEROST the ae iiestceMoccecriatsccas +42.0 +82.6 —78.3 160.9
Other MDimtomse sees seek cee: +40.9 ays —81.8 109.1
Brachionus satanicus.............cescccee +12.8 +21.9 —23.8 45.7
PPEGNTO TI ery te ee ea MUS Bee +70.0 +81.8 —80.7 162.5
(G2 76 le) cota ED Se Ace eee a +60.0 +60.0 — 100.0 160.0
Taife rye LV Dae A SW BEE Or +66.6 +122.2 —77.7 199.9

Table XIII shows the results of the analyses of series G. Here the
total amount of plankton varies considerably since the variation of the
different species is more or less parallel. The chemicals are quite con-
stant. A summary of the variation of the different plankton forms is
shown in Table XIV.

Series H. Since in the previous collections comparatively few crus-
taceans had been obtained, it was decided to collect larger samples. On
October 15, 1915, four samples were therefore collected from approxi-
mately the same points as those of series C during clear and almost per-
fectly calm weather. Two liters of each sample were filtered thru fine
bolting cloth. This allowed the diatoms and some of the algae to pass
thru but retained all the crustaceans and rotifers. This was determined

TABLE XIV
PERCENT OF VARIATION FROM MEAN OF SERIES G

ee oe

Mean Maximum | Minimum Range

DS Gon br Fake F: Wane saopoo Na a ae a ana +26.4 +50.0 —47.4 97.4

Other/Al wae wenr ee erece ce seccwsh tees + 37.0 +74.0 —47.1 121.1
CHRELOCETOS reer e see eae -seoncace areas +50.8 +51.6 —81.3 132.9
Other Diatoms; ee ee estes +36.8 +57.9 —36.8 94.7
Brachionus satamicus...........0e0cee + 34.7 +47.8 —62.5 110.3
Wedalion so: eka ee eee ees rateo ces +78.2 +153.7 —86.0 239.7
Cyclops cc... tera eee aera +35.1 +65.2 —43.5 108.7
Diaptomius):. 8... o sy atiaes cnesconaieces +50.0 +100.0 —50.0 150.0

LES To) 1 Raa Anite oe preter p nee Sy secre +63.6 +78.2 —92.7 170.9

TABLE XIII
DATE FOR SERIES G
(Abbreviations as in previous tables)

Sample 2 Sample 4 Sample 6 Sample 8

Amt. % var. Amt. % var. Amt. % var. Amt. % var. Mean
ORV lenin. cea rate 6.0 +1.6 6.2 +1.6 6.2 +1.6 6.1 0.0 6.1
ree vAMIMO Dl Asesscnnvererten: 0.02 0.0 0.02 0.0 0.02 0.0 0.02 0.0 0.02
Albuminoid Ammonia............ 0.40 — 20.0 0.40 — 20.0 0.60 | -+20.0 0.60 | +20.0 0.50
CO; ion... "i 219 —3.9 217 —4.8 199 —12.7 Di +21.5 228
HCO; ion 618 —0.5 622 —0,2 645 +3.9 601 —3.2 621
Nodularia 78,000 +2.6 | 114,000 +50.0 | 40,000 —47.4 | 72,000 —5.3 76,000
GUEG DOANE AG oc, is caseiedssnetivscecntae 104,000 —47.1 | 342,000 +74.0 | 172,000 —12.5 | 168,000 —14.5 | 196,500
(CHRETOCETOSEs asa ateiiescscsesees 102,000 —20.3 | 192,000 +50.0 | 24,000 —81.3 | 194,000 +51.6 | 128,000
OPHEE IDIAGOMS, «ct savscnrestvercn: 30,000 +57.9 | 12,000 —36.8 | 12,000 —36.8 | 22,000 +15.8 19,000
Brachionus satanicus.............. 1,652 +25.6 1,944 +47.8 493 —62.5 eal? —10.9 1,315
Betlalionssenttins ation ean 28 —69.9 236 +153.7 13 —86.0 | . 96 +3.2 93
CANE) 0) ee rn 52 —43,.5 96 +4.3 67 —27.2 152 +65.2 92
MOS er LOM tieec sevavesesssevieresrtcoses 32 +100.0 8 —50.0 8 —50.0 16 0.0 16
NERC Urea tarkresteviesevisivise+anes 72 —34.5 164 +49.1 8 —92.7 196 +78.2 110

254 MOBERG

by an examination of the filtrate. No chemical analysis was made and
the plants were not counted. The results are shown in Table XV.
TABLE XV
DATA FOR SERIES H

(Abbreviations as in previous tables)

Sample 1 Sample 2 Sample 3 Sample 4

Depth see eo 4.6, —10.7) 5.2) +1.0) 5.3) +2.9) 5.5] +6.8)* 5.15

Temperature.......... 1s) ea ee pol |Aia: F| Me ee D:D) Sete se 9.0). ech eee
Mona besten 124 |+129.6| 23 | —57.4| 47 | —13.0} 22 | —59.3 54
Diaptomu............... 0 |—100.0} 4 | —48.4| 27 |+248.4) 0 |—100.0} 7.75
<Syclops.c eso 2 | —46.7| 8 |}+113.3) 3 | —20.0) 2 | —46.7| 3.75
Brachionus satani-

CUS eee nse 600 |+222.6} 38 | —79.6| 64 | —65.6| 43 | —76.9} 186.0
Pedalion.................. 8 | —38.5} 21 | +61.5) 6 | —53.8} 16 | +23.1] 13.0

At point 1 where the depth is the least the temperature is about one-
half degree higher than at the other points. At this point, also, Brach-
ionus satanicus and Moina, the most abundant animals, are present in
great numbers. Whether this “swarm” was caused by the slight dif-
ference in depth and temperature one cannot say, but it is probably only
a coincidence, since farther on, where the water was deeper, another swarm
of Moina was noticed. The summary is given in Table XVI.

TABLE XVI

PERCENT OF VARIATION FROM MEAN OF SERIES H

Mean Maximum | Minimum Range
Brachionus satanicus...........::00c0 +111.2 +222.6 —79.6 302.2
Ped aligncrere etree erent +44,2 +61.5 —53.8 115.3
Wiis Ro yb at: Hye So tt ant ay a ens ane cee + 64.8 +129.6 —59.3 188.9
(Cy Clone ate eres ote regries ten +56.9 +113.3 —46.7 160.0
Diaptonius.s. sco e ee ste eee: +124.2 +2484 — 100.0 348.4

The distribution of the two sexes of Moina in this series is interesting.
On the whole the females are in the majority except at point 2 where the
males are about twice as many. At the point immediately preceding,

HORIZONTAL DISTRIBUTION OF PLANKTON 255

where a considerable number of Moina is found, the males are totally
absent. The following is the detailed distribution:

Point 1 0 males per liter 124 females per liter.
) 2 15 ”? 9 ”) (a ) ” ”?
”? 3 5 ” ”? ”? 42 ) ”? ”)
”) 4 2 2? PP ) 20 ) ? ?

Series I. In order to obtain still larger volumes of water a series of
three samples was collected on August 17, 1916, with the plankton pump
at a depth of about two and a half meters from an anchored boat. The
weather was calm and when the two last samples were taken the sun
went under a thin cloud, which hardly had any effect on the light. Each
sample consisted of five gallons (18926 cc.) and was concentrated to 18.9
cc. All the animals in five cubic centimeters of the concentrated sample
were counted and the results are shown in table XVII.

TABLE XVII
DATA FOR SERIES I

(Abbreviations as in previous tables

Sample 1 Sample 2 Sample 3

Amt.| % var.| Amt. |% var.| Amt.|% var.| Mean

FSP IATIC IIIA eetctesctesce eee 3 | +30 1 | —56.5 3 | +30 2.3
ped alione resi 8 hae ate: 73 | —12 78 —6 98 | +18.1} 83.0
Brachionus satanicus..............0.026- 54 +4 54 +4 48 | — 8 52.0
JUSS) IV i ee eae ee 55 | +10 50 0 45 | —10 50.0
Datos ee i ee 7 | +40 3 | —40 5 0 5.0
CV ClOp serra att Ba 6 | +81.8) 2 | —394; 2 | —39.4 3.3
VIO a ee es Ae 0 |—100 3 | +30 4 | +73.9 2s

In these collections the adult crustaceans are comparatively few, but
since a large volume was collected and counted the results may be con-
sidered reliable. In the case of the adult crustaceans the variation is
large while in the case of the Nauplii and the rotifers it is rather small.
The summary of the variation is shown in Table XVIII.

256 MOBERG

TABLE XVIII

PERCENT OF VARIATION FROM MEAN OF SERIES I

Pedaliontee ee ee +12 +18.1 —12 30.1
Brachionus Satanicus:....2.05....-ece aE SS +4 — 8 12.0
IN ciarpolitie ete ee te ee can ce, Sea +6.7 +10 —10 20.0
Mia INR eee Ce ek + 26.7 +40 —40 80.0
CSUGIGIIS en eat oe ee aie: SEGRNS. +81.8 —39.4 121.2
NY CaS be EE ge a es Oa are +68 +73.9 —100 173.9

Series J. A series of five surface samples were collected on August
27, 1916, from points lying about 100 meters apart in the center of Creel
Bay as shown on the map. The lake was perfectly still and had been so
for over twelve hours. The volume of each sample and the portions
counted were the same as in series I. Theresults obtained are shownin
Table XIX.

These results confirm those already obtained. Diaptomus and Moina
are rather numerous, still the variation shown is large. The Nauplii
and Rotifera are not as abundant as usual but their distribution is rather
uniform. The temperature and the depth are almost constant, and
from previous work it may be concluded that the chemicals vary but
slightly. Table XX shows the summary of the variation.

TABLE XX

PERCENT OF VARIATION FROM MEAN OF SERIES J

Mean Maximum | Minimum Range

— | | — |

Pedalion: 20 ool eee teh eens +31.6 +48.4 —45.2 93.6
Brachionus satanicus...-sc ee +31.1 +55.5 —33.3 88.8
INatupliti: i) .2.)c. etcsts ccc eeseeeeees ap EVIL +24.5 —35.9 60.4
ADEA CORIUS: : 0. Sasene ene +50.2 +112.8 —71.8 184.6

TABLE XIX
DATA FOR SERIES J
(Abbreviations as in previous tables)

eee

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

Amt. | %var. | Amt. -| %var. | Amt. | %var. | Amt. | %var. | Amt. % var. |Mean

ABT ANGINA tat cwrcicieniiantnc LE es erenestes OPS hivcralanitae Oia earners Os ena O°: laa tenn eres
Pe eOa Monat warincteu ake. 17 —45.2 | * 41 +32.3 24 —22.6 28 —9.7 46 +48.4 31

258 MOBERG

DISCUSSION OF RESULTS

As far as one can tell from the results the total amount of plankton
seems to have a quite uniform distribution, except in a few cases where
most of the organisms occur in large or small numbers in a certain place
or at a certain time. No correlation is shown between the animals and
the plant or chemical constituents. Without further investigation it
cannot be said, however, that plankton animals are not in any way
affected by the amount of phytoplankton or dissolved chemicals. The
variation in the latter may be due largely to experimental error. It
is probable that if there are variations in their distribution they are
small and not likely to cause movements of the plankton animals. The
depth and the tmeperature are always nearly constant for the whole
series and the small variations that occur do not show any effect on the
distribution of the plankton. All of the plankton species show an uneven
distribution on all occasions, even when the individuals of a certain spe-
cies are very numerous. Table XXI gives a summary of the variation
of the individual species. The figures are obtained by taking the aver-
age of the mean per cent of variation from the mean and of the range of
variation of all the series in which the particular species is present in
numbers large enough to be considered. The table shows that the crus-
taceans have the least uniform horizontal distribution, the mean varia-

TABLE XXI

MEAN VALUE AND RANGE OF “‘ PERCENT OF VARIATION FROM MEAN” OF
ALL THE SERIES

No. of series Mean Range

averaged
1a LE 9 a re Sere 5 + 29.8 93.6
MphersAlgaes 32. eiu vetoes eeaere 5 +221 70.1
Chaetoceros iret eats eta teercces = +42.5 127.0
Others atonis ee eee eeese: 3 +49.1 149.5
Brachionus satanicus............0ccee 9 +49.8 148.2
Brachionus miilleri.....................-.-+- 4 +48.6 142.6
Pedalion®. scsi ee ines 9 +40.7 117.1
Asplamchnias jis. tcchitee serene 1 sey hey 218.7
Mong: 34.08 43 coec em ese 4 +55.3 184.9
Cyclone. oo ieee chee 7 +48.5 136.0
DiAPtOMUs + fet ee ieee steers 6 +54.6 172.8
PS pli... 5 oss. cceeee eee 8 +40.3 125.8

HISTORICAL DISTRIBUTION OF PLANKTON 259

tion for the adults of the three species being about +53%. For the four
species of rotifers the average is +46%, and that of the plants is about
+30%. As has been stated before the experimental error in case of the
plants is probably large, and may account for a great part, or all, of
the variation found. For the rotifers and the crustaceans the exper-
imental error is much smaller, owing to the large portion of the sample
counted and to the fact that the animals are more easily retained in
filtering, the variation found consequently being due almost entirely to
the uneven distribution. It is not probable that the small number of
individuals that are sometimes found is sufficient to explain many of the
variations, since in series H, where some of the species are very numerous,
the variations are above the average.’

Direct observations were also made by examining the water surface
for aggregates of animals. During the summer of 1915, before Moina
appeared, crustaceans were frequently not seen for large areas but when
they occured there were usually several together. On two occasions
in August and September Moinas were present in the open water a few
meters from the shore, so numerous that they could be seen from a dis-
tance of several meters. These “swarms” covered an area of about one-
half to one meter in diameter while the surrounding water was almost free
from these animals. Also when series H was collected two aggregates of
crustaceans, chiefly Moina, were noticed in the center of Creel Bay.
Here, however, the areas were larger than, and not as distinct as those
near theshore. During the summer of 1916, such aggregates were seen on
several occasions by Dr. Young and by the writer, both near the shore
and out in the open water.®

COMPARISONS OF THE RESULTS WITH THOSE OBTAINED BY
OTHER INVESTIGATORS

In every case that is known to the writer the collections for the study
of the horizontal distribution of the plankton have been made with a
plankton net and, with but few exceptions, have concerned only the total
amount of plankton. These results usually show a small variation but
tell nothing of the distribution of the individual species. As far as one
can tell from the data for Devils Lake the distribution of the plankton as
a whole in that lake seems to be similar to the distribution elsewhere.

260 MOBERG

A study of the subject was made on Lake St. Clair, Michigan, by
Reighard in 1893.° He collected fourteen series, each consisting of two
(in one case of three) successive hauls made in the same place. The
volume of plankton per square meter of surface was then determined
and the average amount in each series computed. In figuring the per-
centages of variation of the different catches from the average Reighard
uses the volume of each catch as a basis while in this paper the average
of the series is used as a basis. In order to make the results comparable
the latter method has been applied to Reighard’s data. For all but one
series the plus and minus variations, and consequently also the mean
variation, are the same since there are only two collections. Table XXII
gives the plus or minus variation for each series as calculated from
Reighard’s data.

TABLE XXII
VARIATION PERCENTAGES OBTAINED FROM REIGHARD’S DATA
Series II Variation from mean in percent 30:8
” Ill ” ” ” ”? ”? + 17.0
” IV ” ”? ” 3”? ” 0.0
3) V ”? ” ” ” ” + 13.9
” VI ”? ” ” ” ” +14.6
” VII ” ” ” ” ” 0.0
” VIII 2? ”? ”) ” ” +5.4
” IDS< 9 ” ” ”) ”> + Ea |
” xX ” ” ” ” ” 0.0
” XIII ” ” ” ” ” +9.5
” XIV ” ” ” ” ” +4.3
”? Nay ” ” ” ” ” + 14.7
” DOA L 99 ” ” ” »” + 10.3
” XVIII ” ”? ” ” ” + 8.4
Average value for all the series Ys i +9.4

In figuring the results by Reighard’s method the variations of series
II of the above series become +23.1% and —43.1%. The last variation
is much greater than any other and Reighard states that it “is possibly
sufficient to be referable to a ‘swarm.’ ”

Similarly Apstein” studied the distribution in some of the German
lakes by collecting thirty-one series, each of from two to five hauls. All
the catches of a series were taken from equal depths from different parts
of a lake. The catches (eighty in all) were undoubtedly all obtained
from the pelagic zone at equal depths, but it is probable that the depth

HORIZONTAL DISTRIBUTION OF PLANKTON 261

of the lake differed at the different stations. The mean value of the
variation from the mean of all the series is found to be +5.5%, corre-
sponding to +9.4% for Reighard’s data. The highest variation found
by Apstein is +22.8%.

Apstein also counted the individual species in three series, (one of
three and two of two catches) and reports some data published by Za-
charias in 1895. From Apstein’s data I computed the percentages for
Diaptomus and Cyclops of two of the series, the catches of the third
being made in widely different parts of the lake. Tables XXIII and
XXIV give Apstein’s figures together with the percentages.

TABLE XXIII
RESULTS OBTAINED FROM APSTEIN’S DATA FOR DOBERSDORFER SEE

(Amt.=number per cubic meter. % var.=variation from the mean in percent)

Sample 27a Sample 27e Sample 27c

Amt. | %var.| Amt. | %var.| Amt. | %var.| Mean

CyelOpss..ie ecg: 122,088 | +6.7 | 93,024 | —18.6 | 128,016 | +11.9 | 114,377
Diaptomu........... 328,320 | —7.6 } 198,208 | —44.5 | 539,947 | +51.8 | 355,492
TABLE XXIV

RESULTS OBTAINED FROM APSTEIN’S DATA FOR GR. PLONER SEE

(% var.=percent of variation from mean)

No. pr. catch | % var.| No. pr. catch | % var. | Mean

Wy clopsiie es ke eos 887 —1.5 915 +1.5 901

Diaptomu...................+ 26 —13.3 34 +13.3 30
(Natale O88 28s 372 4-13.1 286 —13.1 329

In Dobersdorfer See Cyclops shows a mean variation of + 12.4% anda
range of 30.5%. For Diaptomus the figures are: +34.5% and 96.0%.
In the case of Gr. Pliner See the variation for each catch and the mean
variation are the same since there are only two series. The percentages
are much lower than those for Dobersdorfer See.

262 MOBERG

In the series collected by Zacharias Hyalodaphnia has a mean varia-
tion of +7% and a range of 15.7%, and the copepods a mean of +5.9%
and a range of 15.1%. (Table XXV). These figures, as well as those of
Apstein, are much lower than the Devils Lake figures, and correspond
more nearly with those obtained by Reighard and by Apstein for the total
amount of plankton.

TABLE XXV
RESULTS OBTAINED FROM ZACHARIAS’ DATA

Amt.=number per volume. % var.=variation from mean)

Schlossgarten Alesborg Rott’s Gart’n

Amt. | % var. Amt. | % var.| Mean
Hyalodaphnia................... 630 | +10.5 540 —5,2 570
Copepodaes sok. 720 —8.9 810 +2.5 790
BOSMINAS Wee See ee 150 0.0 150 0.0 150

Kofoid" tested the longitudinal distribution of the plankton in Illinois
River by making “a series of ten catches in immediate succession from a
boat anchored in mid-channel.’”’ The current was flowing at the rate of
nearly two miles an hour, and the time required for collecting was about
two hours. The catches therefore represent plankton taken from a body
of water about three milesin length. From the centrifuged material the
volume of plankton per cubic meter was computed and the following
percentages were obtained: mean +3.6%, maximum +8.6%, minimum
—5.5%, and the range 14.1%. Kofoid’s results thus show a smaller
variation than those of Reighard and of Apstein, but cannot very well be
compared to samples obtained from a lake, especially during calm weather.

The distribution of Daphnia hyalina in Lake Geneva was studied by
Gandolfi-Hornyold and Almeroth” during the summer of 1913. Vertical
hauls were made with a net and the number of individuals in each catch
were counted. All the catches taken from the same depth on the same
day were then compared, regardless of the location and the depth of the
lake at the place where the collections were made. From some data
given by the authors the percentages were computed and are shown
in Table XXVI.

HORIZONTAL DISTRIUBTION OF PLANKTON 263

TABLE XXVI
RESULTS OBTAINED FROM Data GIVEN IN GANDOLFI-HoRNYOLD AND ALMEROTH’S
TABELLE I
Depth of |Depth of Naber per Mean % var. Mean Range
Lake catch eiech from mean _ |lvar. in %} of var.
in m. in m. in %
40 yl by aa fo (E —87.5 E}
45 E E 9 E E —62.5 E|
15 E 10-0 E 41 E 24 E +70.8 E +89.2 241.7
10:5, E Bi ask E —70.8 E
iE) Liey Gigs | | E+4+154.2 Ej
45 —E E 185 E (E -19.7 E|
E 20-0 E E 230:5 | E E/ +19.7 39.4
40 E E 2/6 E) E +19.7 E)
40 E| | E 236 E | (E —17.0E |
a 30-0 |, E E ( 284.5 |, E ei +17.0 34.0
40 E | = 333. B | E +17.0 E}

The catches taken by hauling the net from a depth of ten meters show a
large variation, but the percentages decrease as the depth and the num-
ber of organisms increase. In some cases the depth of the lake varies
considerably but this does not seem to have any effect on the number of
organisms.

“Swarms” or aggregates, similar to those seen in Devils Lake, have
been discussed several times by different investigators, e.g. by Huitfeldt-
Kaas,® Reighard,* and Ward. The aggregates usually consist of
Cladocera and in many cases they have been observed near the shore,
but occasionally in free water. No great consideration is given them,
however, since they are supposed to occur but seldom and therefore do
not greatly effect the results of quantitative plankton studies. The
comparatively irregular distribution shown by the zooplankton in Devils
Lake is in all probability very constant since about the same results
were obtained for all the series. The methods used are quite thoro and
no large error is possible. It is significant, also, that both the Sedgwick-
Rafter method and the pump method give about the same variation per-
centages in many cases. It is hardly conceivable that the organisms in
Devils Lake should have a more irregular distribution than those else-
where, but no work has been done that can be exactly compared to that

264 MOBERG

done on Devils Lake. Gandolfi-Hornyold and Almeroth’s results show
a large variation of Daphnia hyalina but the distances between the col-
lecting stations were probably great and there were differences in depth
and probably also in temperature. Apstein’s and Zacharias’ counts of
the individual species show a distribution quite similar to that usually
found for the total amount of plankton.* Catches made with a net, as
in the above mentioned cases, represent the number of individuals in a
vertical column of water, and it is possible that the vertical distribution
for the different catches differed altho the total amount for the entire
column differed but slightly from that of another column. If this were
the case the horizontal distribution for the different levels would differ.
The collections made in Devils Lake to test the vertical distribution (see
table I) show a large difference in the number of animals of a column of
water between two periods of collecting. Moreover when a large volume
of water is collected, especially with a net, the differences in distribution
tend to be reduced, since by this method several thickly populated por-
tions of water may be included, while on the other hand the small sample
usually collected for filtration in the Sedgwick-Rafter method may be
obtained entirely either from a volume of water containing a “swarm”
or from one where the organisms are scarce. This method is conse-
quently the more precise for studying the local distribution of plankton
forms.

Nor can the distribution of the total amount of plankton in Devils
Lake be compared to that in other lakes, since the data for the former
concern the individual species only. In the majority of cases it appears,
however, that some species make up in volume or weight for the diffi-
ciency caused by others. Since the main portion of plankton usually
consists of algae a large variation of the animals does not greatly effect
the distribution of the plankton as a whole.

CONCLUSIONS

From the results obtained by the study of the horizontal distribution
of the plankton in Devils Lake the following conclusions may be drawn:

(1) The zooplankton in Devils Lake shows a great irregularity in
horizontal distribution, and this irregularity cannot be correlated with
any variations in amount of phytoplankton or in the chemical and physi-
cal environment. It is more likely due to the habit of swarming among
plankton animals, due perhaps to a social instinct, similar to that found

HORIZONTAL DISTRIBUTION OF POANKTON 265

in many other groups of the animal kingdom. Plankton swarms are at
times visible, even at considerable distances, to the naked eye.

(2) With larger samples (19 litres) the variations tend to be reduced,
but even here they are at times greater than in the smaller ones (% litre).

(3) Similar, tho in general smaller variations have been found by
other workers, but no exact comparison with their results is possible,
since their methods have been different.

(4) Definite conclusions regarding the distribution of the phytoplank-
ton can not be drawn, owing to the inaccuracy in the method of its emun-
eration. In general, uowever, it appears to be more uniformly distributed
than the zooplankton.

(5) These variations invalidate the usual assumption that a given
sample of water is representative of a large area, at least in respect to
its animal inhabitants, and necessitate the collection of large numbers of
samples before definite conclusions regarding their distribution or move-
ment can be drawn.

NOTES

1Qwing to Mr. Moberg’s absence on military duty in France, I have taken the
liberty of editing his paper, adding some observations as footnotes and making a few
changes in the text. The conclusions are mainly my own, but apart form these, and
a few other minor alterations, the paper is his. R.T. Young.

2 See Whipple “The Microscopy of Drinking Water.” 1914, pp. 28 et seq.

8 The chemical analyses were made by Dr. Fred H. Heath of the University of
North Dakota.

4 Whipple, /.c. p. 42.

Whipple, /.c. p. 41.

6 In the Journal of the Quekett Microscopical Club, Vol. XI, pp. 373-4, Rousselet
has described a new species of Brachionus from Devils Lake, under the name of spatio-
sus. As this form closely resembles B. miilleri, and numerous transitional forms occur,
it is here included in the latter species. R.T. Y.

7 In series H, only two litres of water were taken. In series I and J, in which 19
litres were taken, the variations are seen in general to be smaller than the average, as is
to be expected. (Compare tables XVIII and XX and XXI) Even here, however,
some of the variations exceed the average, while others are almost as great. (Compare
the range of 184.6 for Diaptomus in series J, table XX, with the average for this genus
of 172.8 in table XXT; Cyclops 121.2 in series I, table XVIII, with the average, 136.9
in table XXI, and Moina, 173.9 in series I with the average of 184.9 in table X XI.)
In general, the more numerous the individuals of a species, the smaller the variations
in their number. This also is to be expected. The variations in the phytoplankton

266 MOBERG

are probably partly attributable, as Mr. Moberg has stated, to experimental error.
In part they are probably also due to chance variations in distribution. For example,
in one case in which Nodularia was exceedingly abundant, I observed it clumped to-
gether in numerous small patches. If one or two of such masses happened to be in-
cluded in a 500 cc sample, while another sample was free from them, they would readily
explain the observed differences. Many of the variations in the zooplankton may also
probably be due to chance, especially in those series where only 500 cc of water were
filtered. Even so they indicate the difficulty, if not impossibility of obtaining
reliable results by the Sedgwick-Rafter method, in the case at least of the zooplankton.

Such an assumption is, however, wholly inadequate to explain such a variation aS
is shown by Brachionus satanicus in samples 3 and 4, ser. D. table VII, in one of which
380 individuals were present in 500 cc, while another contained 0. Similarly 124
Pedalion were present in one of these samples and none in another. Vice versa,
sample 3, in which no rotifers whatever occurred, contained 12 Cyclops, while sample
4, in which rotifers were abundant contained only 4 Cyclops. The comparatively
few Cyclops present can hardly have determined the difference in number of the roti-
fers. The two samples were taken at points only about 200 metres apart in the main
body of the lake which is roughly 15 x 7 Km in extent. The day was clear with but
little wind and the physical and chemical conditions at the two stations were virtually
identical, as may be seenin table VII. An explanation of such variations, as due either
to chance or experimental error is, in my opinion, wholly excluded.

For further evidence of a similar character see Diaptomus, samples 2 and 4, ser.
J, table XIX, in which 19 litres were sampled; Moina and Brachionus satanicus, sam-
ples 1 and 2 and Diaptomus, samples 3 and 4, ser. H, table XV, in which two litres
were sampled; Brachionus Satanicus and Nauplii samples 3 and 4, Ser. E, table IX;
Pedalion, samples 1 and 5 and Nauplii, samples 5 and 7, Ser. F, table XI, and Brachion-
us Satanicus, and Pedalion, samples 4 and 6, and Nauplii, samples 4, 6 and 8, Ser. G,
table XIII.

These conclusions are furthermore supported by direct field observations. (See
FAs) Ree aN

8 The following is from my notebook: ‘‘9-17-17. I notice copepod swarms very
clearly today. In places, usually in streaks, the water is milky with Diaptomus, in
others very few. Occurred at surface. Sunny . . . 9-18-17. I notice numerous
copepod (mostly Moina) swarms in the surface water near shore, these forming streaks
in the water visible plainly at a distance of several feet. I made a collection of one
of these swarms, 500 cc, which I concentrated by filtering thru No. 20 bolting cloth.
Collection made by simply dipping up some of the swarm ina quart jar. . . ”

This collection when concentrated to 30 cc and counted gave approximately
70,000 individuals per litre! This number, moreover, is probably somewhat too low,
owing to a number of the animals adhering to the pipette in transferring to the count-
ing cell) Reda. Yi:

® Reighard “A Biological Examination of Lake St. Clair,” Bulletin of the Mich-
igan Fish Commission, 1894, No. 4.

10 Apstein “Das Siisswasserplankton, Methode and Resultate der quantitative
Untersuchung,” 1896, pp. 51 et seq.

HORIZONTAL DISTRIBUTION OF PLANKTON 267

1 Kofoid “The Plankton of the Illinois River,” Bulletin of the Illinois State
Laboratory of Natural History, 1903, pp. 269 et seq.

12 Gandolfi-Hornyold and Almeroth, “Mitteilungen iiber die Verteilung von
Daphnia hyalina Leydig im Genfer See (Petit Lac), Internat. Revue d. ges. Hydrobiol.
u. Hydrogr., 1915, Bd VII, pp. 426-432.

13 Huitfeldt-Kaas, ‘‘Plankton in Norwegischen Binnenseen,” Biol. Centralblatt,
1898, Bd XVIII, pp. 625 et seq.

14 Reighard, l.c. p. 32 et Seg.

15 Ward “A Biological Examination of Lake Michigan” Bulletin of the Michigan
Fish Commission, 1896, No. 6, pp. 62-64.

16 Tn the case of Cyclops and Diaptomus for Dobersdorfer See, Apstein finds a
somewhat larger variation. The mean for Cyclops is 12.4%, and for Diaptomus
34.5%. For Diaptomus the maximum is +51.8% and the minimum —44.2%, mak-
ing a range of 96%. In the 10-0 meter catches Gandolfi-Hornyold and Almeroth find
a large variation, but in the catches from greater depths it is considerably smaller.

DEPARTMENT OF NOTES AND REVIEWS

It is the purpose, in this department, to present from time to time brief original
notes, both of methods of work and of results, by members of the Society. All mem-
bers are invited to submit such items. In addition to these there will be given a few
brief abstracts of recent work of more general interest to students and teachers. There
will be no attempt to make these abstracts exhaustive. They will illustrate progress
without attempting to define it, and will thus give to the teacher current illustrations,
and to the isolated student suggestions of suitable fields of investigation.—[Editor.]

GENETICS IN RELATION TO AGRICULTURE

Under this title Professors Babcock and Clausen have brought to-
gether in a most valuable way two winning groups of interests. The
development of agriculture as an application of various underlying
sciences has been one of the very creditable outcomes of scientific pro-
gress. And the growth of the educational aspects of agriculture has been
the wonder of modern education, which with the aid of shrewdly used
political appeals has made more than one of our universities the tail to
an agricultural kite. On the other hand, none of the divisions of bio-
logical science approaches that of genetics in the impetus which it has
given in recent years to research. This is true whether we are thinking
primarily of the discovery of new facts or of the theoretical conclusions
to be had from them. If therefore we acquiesce, as we probably must, in
the authors’ statement that no field of science contributes more of eco-
nomic worth than genetics does to the complex called agriculture, we
have a measure of the possibilities of a book on this subject. In the
opinion of the reviewer the book is peculiarly valuable, not merely to
agricultural students for whom it is primarily written, but for teachers
and students of biology everywhere, for the general reader, and for the
breeder. A very rich selection of illustrative material has been made—
much of it from sources not familiar to the general student.

The subject is treated under three heads: Fundamentals; Plant
Breeding; Animal Breeding.

Part 1, dealing with the Fundamentals of genetics comprises fourteen
chapters. Biologists will agree, I believe, that the various hypotheses
have been fairly stated and the pros and cons of the more uncertain ques-
tions justly given. The illustrative material is pertinent and modern.

AMERICAN MICROSCOPICAL SOCIETY 269

Part 2 discusses Plant Breeding in twelve chapters and contains such
representative chapters as, Historical Introduction, Varieties in Plants,
Composition of Plant Populations, Selection, Utilization of Hybrids,
Mutations, Graft Hybrids and other Chimeras, Breeding Plants for
Disease Resistance, Methods of Plant Breeding.

Part 3, Animal Breeding includes thirteen chapters. These run
parallel to those of part 2, with some of peculiar interest added—as for
example, Disease and Related Phenomena in Animal Breeding, Sex
Determination in Animals, Fertility in Animals, and Some Beliefs of
Practical Breeders. The latter deals briefly with the scientifiic grounds
for disbelief in telegony, maternal impression, prepotency, and the like.

The concluding chapter states the grounds for a becoming modesty
in relation both to the quantity and the precision of our present knowl-
edge of animal genetics.

The book contains also a glossary, a list of the literature cited, and an
adequate index. It is richly illustrated with pictures, diagrams, and
tables. It is an attractively made book, and is sure to prove a useful

and satisfying one.

GENETICS IN RELATION TO AGRICULTURE, by E. B. Babcock and R. E. Clausen. Pp. xx +675,
fully illustrated. The McGraw-Hill Book Company, New York, 1918.

NITRATE CELLULOSE AS A SUBSTITUTE FOR CELLOIDIN

Asa result of the war the importation of celloidin has been interrupted
and the microscopist has been compelled to look about for workable,
substitutes. Parlodion has been found to be very satisfactory, and can
be obtained from the Arthur H. Thomas Company, Philadelphia. In this
laboratory, however, we have had such excellent results with nitrate
cellulose (soluble cotton) that I feel justified in calling it to the attention
of other workers. Although never in very general use, soluble cotton as
an embedding medium has been known for some time, and has been used
for a number of years in the laboratory of Dr. Adolf Meyer, John
Hopkins Hospital, as a routine method of embedding. It has two valua-
ble features—the cost is less than any of the other practical celloidin
substitutes, and its preparation is comparatively simple.

Nitrate cellulose is shipped in strong alcohol, and upon reaching the
laboratory is put through the following process: It is washed first in
several changes of 95% alcohol and squeezed nearly dry; then in two
changes of absolute alcohol, after which it is dissolved in equal parts of
absolute alcohol and ether, filtered through absorbent gauze into a flat

270 NOTES AND REVIEWS

dish and placed under a bell jar to evaporate until dry. It is then cut
into thin strips and put into a thermostat for several hours at a tempera-
ture of 37, the door of the thermostate being left ajar to allow for the
escape of the ether fumes. When the chips are thoroughly dry they are
stored in air-tight bottles ready for use. Where haste is necessary the
filtration through gauze may be dispensed with, the cotton being de-
canted as it dissolves and evaporates slowly under a bell jar. The bottles
are then placed in the thermostat under the same conditions as described
above. This, however, is a crude method, useful in ordinary work, but
not to be followed where careful infiltration is desired.

For embedding we use the same technique as for celloidin. Eight
wide mouthed, cork-stoppered bottles are cleansed and thoroughly dried.
The solutions are made up in such a way that each 100 cc. contains 2, 4,
6, etc., up to 16 grammes (by weight) of the soluble cotton. Tissue that
has been thoroughly dehydrated and immersed in equal parts of absolute
alcohol and ether, is then passed through these graded solutions, being
left 24 hoursin each. If the tissue is to be cut immediately it is mounted
on a fibre block and hardened in chloroform or in 80% alcohol.

Nitrate cellulose can be obtained from Maas & Waldstein, New York.

Cuas. H. MILLER

Department of Embryology

Carnegie Institution of Washington

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CopnsenPRor, GEORGE FE, Ph.Ds 719 geo Ve R. F. D. 9, Lawrence, Kas.
COLTON ERMRORD Ss. D 710: Zoological Lab., Univ. of Pa., Philadelphia
CONE MAEBERT lance ne lay ok tytn os. Editorial Staff, “ Lumberman,” Chicago, Ill.
CONGERVALTEEN Cayenne (Sy 28 alo, aL esi P. O. Box 663, East Lansing, Mich.
CONTON, (AMES Johari ce a 717 Hyde St., San Francisco, Cal.
Cooper, ARTHUR R., A.M., 716............000000+ College of Medicine, Univ. Ill., Chicago, Il.
CoRgne ct Univ. [aprary (PROF.'S: Hl. GAGE) o.))60) cscs ccckececesecesteeee Ithaca, N. Y.
Coen! Wi Wel Phi aie ate IO Ra ae dn Dept. Zool., U. of Cal., Berkeley, Cal.
omr GEORGE :, Msi MON i alae So Ae 1001 Main St., Buffalo, N. Y.
RSOVES,) GEORGE: Wis UD a ae a ee Laeeak College View, Nebr.
DARBAKER, LEASuRE KiIng, Ph.D.) MDs 700.00... cescecssaessaeeeeeenie cee

PG NAC CUZ nC RAE ReiSln R RUE AR 7025 Hamilton Ave., Homewood Sta., Pittsburgh, Pa.

AMERICAN MICROSCOPICAL SOCIETY 273

Wages Puce H4 SPD; 719.0 ce University of Florida, Gainesville, Fla.
DreRE EM OLA AVM SUM, 713... ee Bethany College, Lindsborg, Kans.
Pye EE CHARTERS Els IVE Scs lei ee .355 College Ave., Valparaiso, Ind.
IDISBROW, WILETAM'S:, M.D, Ph.iG:, 700.) ...2....2.<s.0203 151 Orchard St., Newark, N. J.
MODGE CARR ODF) Wohi G bar ee LLU Ue ita ley” al TE EN en ier Pawlet, Vt.
DOLBY. EDWARD PE) (OGxer hee eet bah 3613 Woodland Ave., Philadelphia, Pa.
DouBLEDAY, ArtauR Ws M.D 716.0... occ 220 Marlborough St., Boston, Mass.
IDRESCHER AWE (OMe tes re Care Bausch & Lomb Opt. Co., Rochester, N. Y.
IDGBES HME wasy ALBERT) 216282 Con 20 ik Sian is eh ls A NEE, CART cae eae Ransom, Kas.
DUNCAN PROF BeEN: eho i Ores e meee ee cee So. Methodist Univ., Dallas, Tex.
EDMONDSON, CHARLES) Hi. "Ph De 15 ee lieecs sess 1360 Alder St., Eugene, Ore.
SBD 5 VEEEEOND Wie Lilie eee SAUD eRe Lee en CTS. Leen di eeasdloen State College, Pa.
IBDDVA GAMUT L Awe ye ite eee ese in LN ee eee cee. Tower Hill, Ill.
INGGTESTOND EDs Reon IVICAGs Gere Rapes De Marietta College, Marietta, Ohio
IGENMANN:) PROF?) CHW OSM Benen se 630 Atwater Ave., Bloomington, Ind
DERIOnn a TRANKG Re MyitAe cl oym inary cee es ol hue unt A Tee eal Wilmington, Ohio
LOUD ested Fano Les vs (OL i) el 9 gs BR ge ee 1109 13th St., Boulder, Colo.
PGROD PEROP MORTON i. MEAL IMES.{ (O80 1c Neenah cee he Tees ce Seer eae
5 cee aed ae StS ae APE Go Seat BS ARO Ao oR University of Montana, Missoula, Mont.
EsSENBERG, Mrs. CHRIstINE, M.S., 716.............0...... Scripps Institute, La Jolla, Cal.
ISTER EV CALVIN Oo lo bse tee, Bau uak Se eee Occidental College, Los Angeles, Cal.
Bere) orn We MDS MES... BReMeSe) 790) ae) cre ee Se
ener tad Sith Hace a PLO RA he ok cL Guy’s Hospital, London, E. C., England
BARTOW SPROFD WelGs Me Se ci ae eee 24 Quincy St., Cambridge, Mass.
ATT CHR OR! EVV seb teyes HIVES: pail Dis hee 9k oa geeks OMe epee Gainesville, Fla.
FEttows, CHAS. S., F.R.MLS., ’83................ 107 Cham. of Comm., Minneapolis, Minn.
FERGUSON, MARGARET C., Ph.D., 711.000.000.000... Botanical Dept., Wellesley, Mass.
FERNANDEZ, Fr. MANUEL, B.S., 716............ San Juan de Latran College, Manila, P. I.
EN DEAY. Mit ING Ce. VALMI S715 2.02 soe a bana Park College, Parkville, Mo.
IES CHIR AN ATER 1 ZT ce atte ies ey tole ae a he Be shee Box 1608, Milwaukee, Wis.
Patz RANDOLPH) SRAVMOND (Bp OR ME Ss.) dies ots eee eee reid eee ete oo cae ee
1 cap MAS AED SOE MIDE. Nee OME TEBE KHER State Laboratory of Hygiene, Trenton, N. J.
cing: JAMES) Mil" MD 1, er oe eae Stoneleigh Court, Washngton, D. C.
BROOME I iis25 (VED EOD bee ore dese e eate 202 S. Thirty-first Ave., Omaha, Neb..
Boveran \WiaiaraM VMS, 16. aa sccteae 707 Coleman St., Easton, Pa.
eM ES SEW: WE) Pht). OS... Silas wh ikon: 52 Vernon St., Hartford, Conn,
GSERTED Lalor eee Ie Millahhe hoary 2659 California St., San Francisco, Cal.
GAGE Pape SIMON EE 5B.) B20 abe ek 4 South Ave., Ithaca, N. Y.
GATrOwAy Eprom, i.) Wa AML PRD, Ot a ey Beloit, Wis.
GARBETSON PUGENE YS Tate oe 428 Fargo Ave., Buffalo, N. Y.
GernSMrMan Ge We mrs hy LL eee ee te es eae Bryan, Tex.
OWEN) WR SCTS Ea ER ee Ah re R. D. 1. Box 14, Exeter, N. H.
Granny, Coarurs Wi Mo AT elles 447 W. 14th St., New York City
GRAHAM, Jonw VouNG PR Da TA ke University, Alabama

GRAY, (WILTAMUCARYIN Ia) tii ul ee Lock Box 233, Tama, lowa

274 LIST OF MEMBERS

GRIFFIN, LAWRENCE Bi; C1352 ine ntscee University of Pittsburg, Pittsburg, Pa.
Gurprrtet, Joun EPR. bee eee A. & M. College, Stillwater, Okla.
Gover, Messer Fy Paella en University of Wisconsin, Madison, Wis.
HAGELSTEIN; ROBERT 1G 00.5 o ee see Seed erence elena Minneola, Nassau Co., N. Y.
HAGUE) PEORENCE AU Mie OU eS ue ee ee lal one Nat. Hist. Bldg., Urbana, Ill.
ALT, GREG REGORV UB YAe yi eee ath ee a Milton College, Milton, Wis.
HANCE MROBERT lL js sAstilige cere eee: Zool. Lab., U. of Pa., Philadelphia, Pa.
TANKINSONG: 7) dls PALS W703 te aes er UES Bh arch A en mR Charleston, Ill.
VANNAPIVIARGARTT is.) ALMics 1G /ee ie erate eee Station A., Lincoln, Nebr.
FIANSEN SF AMES ul Snail a hlasne teeta St. Johns Univ., Collegeville, Minn.
|B Pao) gy ] Dic(eroi spo & Ls Beye Rs i RN ky nee ee neh eee oi ta 8 1860 12th Ave., Moline, Ill.
ITARMAN, OMAR Veil. C13%2 fs ces Kansas State Agr. College, Manhattan, Kansas
HAYDEN. JHORACE Hipwint JR. (14. oe. ne kann College Station, Texas
Je bona) 1 Dey 11D) 2 el Dee 0 ode ede ee Wash. State College, Pullman, Wash.
EEA Ter OR ON PEUR AN KSGLN Se VIS Cool Osseo se ta «50k ose se rceodeausese teas South Beach, Ore.
HEIMBURGER, HARRY V., A.B., 714...0....cccccsc000. 1625 Wesley Ave., St. Paul, Minn.
EIENDERSONS (WVIDETA MA ped: fo pep tees eae oy eecasctet Millikin Univ., Decatur, Il.
ELTETON] WTEDTAM AS mB) cil id een center eer eer eee, eae ee Claremont, Cal.
NATTSSONG, BIROWa 0D) OB tay Oe ieee coco set ce dct eee RE eo bee ara Madison, So. Dak.
HIrcHIns, ATERED) B.D iyi, cence estore: 2 Dwight Block, Binghamton, N. Y.
Hyorre, Lupyie Ge A2e ee eo Meadowdale, Snohomish County, Washington
Hoty Cross CoLiteGE, PROFESSOR OF BIOLOGY.............ccccceceeeeees Worcester, Mass.
TIOSKANS, SNVMiSY 1 Ope ee onal Eh BEL Ly eA 49 6th St., LaGrange, Ill.
Howarp, Rospert NEssit, 712........ Ookiep, Namaqualand, Cape Province, S. Africa
HOWLAND, Henry Ri, ALM. 798 .f.2.c.ssccsestecenseccsoet 217 Summer St., Buffalo, N. Y.
Ji hofeds aolspg Sy-Wp1 b'day] Soe ial I ped a JRRe a eae fe Grinnell College, Grinnell, Iowa
VES SERED ERIC TH MOL a re ce eek rere et ie 1201 Race St., Philadelphia, Pa.
PERRS SR ROR RG lice ollie pon eae SU ee Univ. of Okla. Norman, Okla.
MPERINISRMES AN: MAN PAD) be eet ech eA a entre Science Hall, Indianola, Ia.
SJ ORINSON SB ii Loe en ot Crake boning een Joplin, Mo., R. F. D. 4-147
JOmnson, (rare P),)D:C.LG.Bs 7hGs o.cttentcases 200 W. 72nd St., New York City
JOHNSON PRANK) S:;) M.D) 203.20 eee 2319 W. 24th St., Los Angeles, Cal.
JJORDANIDPROR. TE Eyed 2 250. Secs. sasctetsostscores University Place, Charlottesville, Va.
EDAv A MmANCE O00 soc. sation cee Biology Bldg., U. of W., Madison, Wis.
RT CAR ETT TARY PS POU ee hisses edhe, ncsteacens oe ees St. Procopius College, Lisle, Ill.
KEELOGC Sa] SE IMIR 21 Boi, coco cosateteens sestecs see: 202 Manchester St., Battle Creek, Mich.
EGER NATIT Se VEORRIS Wale PAS IVE o-oo. tas So ionn.5 eS Uee ose oat oe Bismark, No. Dak.
IGINCATD MDREVOR Aeon ae ne ares: University of Washington, Seattle, Wash.
ISIN GG LN Zeya artnet: eg ots atten res ote ences Centerville, Iowa
ISINGS WIEIIARD WV es hal geeeee te casei ceec P. O. Box 261, New Orleans, La.
Kirscu, Pror. ALEXANDER M., M.G., 716..............-.:-20000++- Notre Dame (Univ.), Ind.
SKGTSENYSMATID 910550550 suc eau le eet eters eace shes sue tet Sut be Sasser Fauld Cea secces 2 cdvaes heheh ae er
Gonos Gee] Oana ao eA eS ot od ee cA 1015 Blondeau St., Keokuk, Ia.
KOrom, CHARLES ASuP HID 09m eerste University of California, Berkeley, Cal.

KeOnZ; SAEs MED sp iO eas ieee ote eaioaces saetiner eerie 32 S. Fourth St., Easton, Pa.

AMERICAN MICROSCOPICAL SOCIETY 275

KReEcKER, FREDERIC H., Ph.D., 715.....0.0000000002... Ohio State University, Columbus, Ohio
Aes RANK We, 8A oe. osc st cess U. S. Naval Hospital, Las Animas, Colorado
Lambert, C. A., 712........ Bank of New South Wales, Warwick, Queensland, Australia
Lanp, WILu1AM JESSE GoaD, Ph.D., ’15........ The University of Chicago, Chicago, Ill.
MOAN Soe Te gibi sce race heed Sechrest a eek ee Univ. of Okla., Norman, Okla.
IVAN EZ 1 EYRUSe Went ACIS WoL Gia: Sire tae Aalst hears ee ee University, Reno, Nev.
ARGH GEORGE RR.) bheDs 11. eee University of Michigan, Ann Arbor, Mich.
Pameamue Wiicsy V4 AC. MD DDS... ROMS. B80) ot oe ke A
Rea ee er ee ON eet Ra 1644 Morse Ave., Rogers Park, Chicago, Ill.
Parewen, Homer “By, MeA.. tes oes oe ste ccses 1226 So. 26th St., Lincoln, Nebr.
GE WIS) EV is Yoiy POR TMEAIND we lie DE ee core en tere nctecs teakseo trae teste University, Va.
ews Mins: KATHERINE Bo 89 5 fo ie tctencistntene te Bellwood Farms, Geneva, N. Y.
Mrewise ie Bardo se So een ee ee Okla Ag. Exp. Sta., Stillwater, Okla.
Big dara ee Sam 21") BN. 5o) 0 Ad 07 uke aR a Nashville, Tenn.
Romp AWOLPH (O22 ence te cache ota 289 Westminster Road, Rochester, N. Y.
LONGFELLOW, ROBERT CAPLES, M.S., M.D, 711....0.000 ee. 1611 22nd St., Toledo, O.
HOW DENA Em GHa Bel Or ee Bele ce etn deeritcenscoveantraee 2120 High St., Denver, Colo.
Levon wHOWARD) Nie MID, 784. 2..2.0.--c8 cts ecseee, 828 N. Wheaton Ave., Wheaton, II.
MacGiiiveay, ALEXANDER D., 712.................... 603 W. Michigan Avenue, Urbana, Ill.
Mack, MarcareT ELIZABETH, A.M. 713...0.000. cece Univ. of Nevada, Reno, Nev.
memenwn.. “CAS. M55 11S 225 oc Se Medical College, U. of I., Chicago, II.
Marn, Grorce Hinry, M.E., 11)... 4c...2dnt-sceiaen.s: 94 Silver St., Waterville, Maine
INVAR SHALE. COLGENS MID). 296) 3.52: cccssc1sticonteissse: 2507 Penn. Ave., Washington, D. C.
GUSSET E709 eo 20 ws 2 «Yl DEN fp a Lane Technical H. S., Chicago, II.
MARSH ATES Wrists, RSI) 129, Aon ye os ee 139 E. Gilman St., Madison, Wis.
MARTLAND, Harrison S., A.B., M.D., 714.000.0000... 1138 Broad St., Newark, N. J.
IMAnrER si D)PhaD 5702 one ck beeen 228 Gratiot Ave., Mt. Clemens, Mich.
May, Henry Gustav, B.S., ’15....Bur. Animal Industry, Zool. Div., Washington, D. C.
MAVIE Wek OVa nO 619 Soe eg aes Wesleyan College, Warrenton, Mo.
MAYWALD; FREDERICK Ji, 702..06.2...-22.s01000-0- 1028 Seventy-second St., Brooklyn, N. Y.
IMCGEENAHANS, (Bnet: MajeclSi 22:8. 0 80 eo Ss ee Manhattan, IIl.
McCrrmry-(Gron Dh s013:2%, ci ror 0 ae) Lyon Co. H. S., Yerington, Nevada
IMG wn fat 2h. 24 eee fee tee be 1118 Marbridge Building, New York
MCKAY ROSEPHS (34.007. US hee errs oleate hcpele Saeed 259 Eighth St., Troy, N. Y.
MeKerver,; Peep L., F.R.M/S.,.’06.22.4.2:5.A:000.3. P. O. Box 210, Penticton, B. C.
McLavcuiin, Atvan R., M.A., ’15........ Med. Dept., U. of So. Cal., Los Angeles, Cal.
Me Riri Orpss Ou VERA; ALB 71 6:22 oocoieccccocccacsacdecssctsTacsvoedtsostse ds Mason City, Nebr.
IMGNVIRIAAMS GIOHNG 140 00..00 0 0s eee a koe) Lock Box 62, Greenwich, Conn.
MErceR, A. CLiIFForD, M.D., F.R.M.S., 82........ 324 Montgomery St., Syracuse, N. Y.
IVER Ey Vier oe ED 290 Fs eae oe lente sh eaesea 200 E. State St., Athens, Ohio
Mercane He Bethe 2a". SE eee EN ee Ps RA EP OE TE Agricultural College, No. Dak.
MertcaLF, Pror. ZENO P., B.A., 712................ Col. A. & M. A., W. Raleigh, N. C.
Minter Crarres res ites iis ot Med. School, John Hopkins U., Baltimore, Md.
Mitier, JOHN AS PhD Hh ReMES: 789) oe 44 Lewis Block, Buffalo, N. Y.

MOCKETE, Hi See 0 temas oe ue Sa en 2302 Sumner St., Lincoln, Nebr.

276 LIST OF MEMBERS

Moopy, RoBeErtT P., M.D.,’07....0.00.0.000.... Hearst Anat. Lab., U. of Cal., Berkeley, Cal.
Morcan, ANNA HAVEn, Ph.D., 716.......0.000.00. Mt. Holyoke Coll., So. Hadley, Mass.
Mvers PRANK sa iii aun En 15 S. Cornwall Place, Ventnor City, N. J.
NESBITT, ROBTAAS TG NOMI ge Nua in ties Box 1171, Sta. A., Lincoln, Nebr.
INOTT,) WHEEEAM IG SWAMI NES Lo SMA) SA, UE aOR Le ea Genoa, Nebr.
NORRIS. PROE.VEARR Ty N\VATIDO ili wue en Cn esas 816 East St., Grinnell, Iowa
INGRTONY CHARTS ENVIR NTL i ar wane, WLM aseatl 118 Lisbon St., Lewiston, Maine
OCTEVEE ZS COS EV BESEISeiD ear diD ae nan ie ONIN a eu a) 1006 N. Union St., Lincoln, Ill.
OszorNn, Pror. HERBERT, M.S., ’05..00.....000c. Ohio State University, Columbus, Ohio
OTT EVAR VaEVING AMMEN OS LAU NUE NLS UO ae Spencer Lens Co., Buffalo, N. Y.
RAG EIRENE MEL BEN TW Nal fe ieeeas i ENR Dasa) aM 528 Stewart Ave., Ithaca, N. Y.
PATER TOMAS CHAME DEY, DSi.) di nC OMAE Tai Media,’ Pai Ry Be. Di
IPAMRI GK WEIR AN KY Ee OMA OM OUNCE INI bl tls 421 Bonfils Bldg., Kansas City, Mo.
PAS AMOR DEN oe Wad Olu Nite) OUV GML Oa MTA MRNA ad P. O. Box 503, Altoona, Pa.
BERRY WiGEORGE (GOSH A MAUMIs Lito wtac UG NUNN a eNO UNL ERNR ee Salem, Virginia
PENNOCK) EDWARD AO unui Reuss 3609 Woodland Ave., Philadelphia, Pa.
PER VAM EOS VOD iA US OAD NbN MU EAL Encampment, Wyoming
PE TERSONAUNTE TSH BREDERTOR Uj i1 aU imne Daan kU pau oa lentaS Box 107, Plainview, Nebr.
Pape MARTIN MeSe i TO weeny 25th St. & California Ave., Omaha, Nebr.
PT) TEST WARD) iy Le Mee SMa eee Brandhock, Gerrard’s Cross, Bucks, England
PEACE ADAM Teh TS Ay ect ume t acu TE Re eRe State Normal Sch., Kalamazoo, Mich.
ProuGcH, HAROLDIE:, AVM 1O6n ae Dept. Biology, Amherst Coll., Amherst, Mass.
PORT MRORINAG RRO AUD IR Ee VM seed aby 204 N. 10th St., Easton, Pa.
POOL RAYMOND UT (ERD Nl Sumiun nat Kune ccy tea Ued tannins Station A., Lincoln, Nebr.
BouNDWROSCOR /AUVIE VPin DOG 0 epi unin Harvard Law School, Cambridge, Mass.
Powers, E.B., A.B., 712...00.0.0.cccscssssss1+s.324 E. Uintah St., Colorado Springs, Colo.
PAR CREO WINES Ss NIRS Mi MARR Lee CAN eal ance 421 Douglas Ave., Kalamazoo, Mich.
PRIENVEROFS OTTO MSM UN IT nee ONe ae 5 and 6 Fedl. Bldg., Laramie, Wyo.
BUR DYANV TEE TAN (Cosi Si) 1 Oieuie uouren uu 3rd & Kilgour Sts., Cincinnati, Ohio
Ooi TIAN MAR van CLAIM Ns Sune T Ue aU ead Wesleyan Col., Macon, Ga.
FRAN TKAINFUNVATT ORS INV De alg le yee alent lars Nels WD Princeton University, Princeton, N. J.
Ransom, BRAyTON H., ’99........ U. S. Bureau of Animal Industry, Washington, D. C.
RN CTOR MH RAN Ken WS EDR Mie Dye mT TMi ene el Naa Luis 227 Fulton St., New York City
REESE, Pror. ALBERT M., Ph.D. (Hop.) ’05......W. Va. Univ., Morgantown, W. Va.
Rae AN el Dae Headquarters 57th Division B. E. F., care G. P. O., London, Eng.
TRICE AAV \ LEELA IE AMIN D7 5 01S YO) OU A ai cy AM aud WA 901 College Avenue, Wheaton, Ill.
RAGHARID Ser AcE, NE TIDY DOS Cece 7 ee Ba Silas Wabash Coll., Crawfordsville, Ind.
RarEy CEC uRmISsIVMIn Ss iil tinue usmle State College Forestry, Syracuse, N. Y.
ROR BRITS BNW TCLS Saliba MOND) als eee Ny oe Rte HU 65 Rose St., Battle Creek, Mich.
ROBERTS PETS LVL imation wil eae iG State Normal School, Cape Girardeau, Mo.
ROBERTS VA Len RMU UNS ALP N GN ysl ti He 460 E. Ohio St., Chicago, Ill.
FROBINSONG) His), Ere) VTC Ata Sy Mnaia GU EKO RMS OU AO MAND! Box 405, Temple, Texas
ROE NG2 CESAR BLY ASME NNAU Nuc eU NYU is, 1032 Elventh St., Boulder, Colo.
ROGERS) WALTER BO Fd Sen Ot) Westminster Col., New Wilmington, Pa.

Ross, LUTHER SHERMAN, S.Mi., 1d iceceledisecsedeoeses 1308 27th St., Des Moines, Iowa

AMERICAN MICROSCOPICAL SOCIETY 277

IROSSTEER EO WARD Mi) AB 80) oh cea Au Sigma Pi House, Athens, Ohio
Tare) 1 ACCOUNT Ni 17 SOE alge eR De BESS AAA ROE RU AN ey Hudson, Ohio
Scott, GEORGE FitmorE, A.M., ’13.......... College City of New York, New York, N. Y.
Sa Tere EARN AYA oo oe AR ACI MLD Se RPC A gC Univ. of Wyo., Laramie, Wyo.
Sysu NRA e Buyd Vigan Boal D Yaad 0 ee DUE Bureau Plant Industry, Washington, D. C.
SEI AR REA AvEs ee Oe etree eae re OME RU a aaa Pe CR 809 Adams St., Bay City, Mich.
SHELDON, JoHN Lewis, Ph.D., ’15................ ....W. Va. Univ., Morgantown, W. Va.
SHIRAMPATSTING ELIN TUB SACRA Sun inek sudan taste DESMA ULL MR UG ys Lake Homer, Minnesota
SHUTDZNCHAGH Sah SOU te we MERU OUR Ala ae IS Seventh St. Docks, Hoboken, N. J.
SIstER Macna, O.S.B., M.A., 716.................. St. Benedict’s College, St. Joseph, Minn.
STEER TD ANB Se heh Olen lee Cis BOON lee een Lake Erie College, Painesville, Ohio
SrOcuM Ke rASH /B)e PID) AMEND feta une aie alerts WLEs | 218 13th St., Toledo, Ohio
SMITH? PROP PRANK: /ASMeN 12ers a! 913 W. California Ave., Urbana, Ill.
Surra,, GILBERT MORGAN. PhDs 15 i 1606 Hoyt St., Madison, Wis.
Sn Did rey [Py Os a 2 Yes AMON ee RA RO ERAN CAS a 131 Carondelet St., New Orleans, La.
Soar, C. D., F.R.MLS., ’07......37 Dryburgh Road, Putney, London, S. W., England
SPAUEDING qin ene Mie bo in ak eo uae 508 W. College Avenue, Bozeman, Mont.
SPURGEON, CHARLES H., A.M., 713.........0.0000.0... 1330 Washington Ave., Springfield, Mo.
STMPETENG Smee tibiae elect Uae Mie aN 15 Whittlesey Ave., New Haven, Conn.
SHEWART DHOMASS:./ MED O29 7 2.0 (iw yy) 18th and Spruce Sts., Philadelphia, Pa.
StEvENS, Pror. H. E., M.S., ’12...... Agricultural Experiment Station, Gainesville, Fla.
STONE GRACEIAL VAM TO et cull Misa UL NH Teachers’ College, New York City
SRUNKARD WHORAGE NVe4 NET eDeied S sO maul ne WAU Aa cased dues taauadenc tatdac soa
RRR aE Slaps IM rR UMi TULA AS DUT UAE LY New York Univ., Univ. Heights, New York City
SURNER SER OR EIB 7S Gay ay Wien tO INULIN NY aN LU ANA UA STUUR Lec UA ait Ames, lowa
SWHZY.) OLE eas iS Vis We East Hall, University of Calif., Berkeley, Calif.
SWINGLE, PROF LEROY) Ds) OGM aera y Univ. of Utah, Salt Lake City, Utah
DACCART Rave REM BW RUA Mince SUT Mia eA UNL ait a Morgantown, N. Car.
AVEOR a OSHPHy Ge NBs Sool Os ule nes Midori ULE MU New York Univ., New York City
ERR ELLER UMAN CoN Ti Dolan 1301 Eighth St., Fort Worth, Tex.
rows, An rar EP! 700). U0 ey Ny W. Washington Sq., Philadelphia, Pa.
SE RNIMIN S's GEORGES 9G s eel vol One 1410 E. Genesee St., Syracuse, N. Y.
INSEE. CRANDOEPHS WORD eBeSu)) al oer un uel mauiy sda Georgetown, Texas
HOD D sam AtES whCx) TBR UACY OME TA iad oe ama ACLAASLULy JURA Da usec LIN SS Boulder, Colo.
BURNIN Rs KOTMDRON 2120 i Eee) pl Nee IVES yaad bali 817 Crescent Place, Chicago, Ill.

College Station, Durham, N. C.
Mass. Inst. Tech., Boston, Mass.
300 N. H. Bldg., Urbana, Ill.
WanWoreubinrraag At 7 Wi Nil cen Cuan! 11 West 45th St., Bayonne, N. J.
Won Kirineneap! ResBe 2980) Nii) Nk University of Arizona, Tucson, Ariz.
WAGNER WED WARD AIA UZ Mion Min 124 Willet St., Jamaica, Long Island
WATTE, FREDERICK © Ud UL lla eecet acetal dnnnnateandssstradeemeantt
said Sth on ae ime Ae Medical Department, Western Reserve Univ., Cleveland, Ohio
Water, Exp R., Ph.D., ’07 University of Nebraska, Lincoln, Neb.
Wa ker, Leva BELLE, 713 Station A, Lincoln, Nebr.

RINDI Y COLLEGE) LIBRARY: (ooo
Boer nC mAbs MALTS. hiv aN
VAN CLEAVE, Hartey J., ’11

278 LIST OF MEMBERS

WARBRICK. Ji Cc P02 6h Roane 2k ia Sak Sper eae 306 E. 43rd St., Chicago, Ill.
Warp, Henry B:, AM. PRD. 872.20 ere! University of Illinois, Urbana, Ill.
WARNER he Ae MED So Phy Gay dijpeete not eee 1213 15th St., Moline, II.
NAGE Ete hcg) arse casey sien yocteeeeereren ee ceaenaee area 1805 Patterson Ave., Roanoke, Va.
WATER WORTH. (ANG, DOM screens 286 Lambton Quay, Wellington, N. Zealand
5 Veo olay cig: Cal O AEG: Reese eee Dhan: 0 ed AM Ee rr Ua The Vivarium, Champaign, Ill.
\i@oase DN ame Sea D Aa ee eae Univ. of Michigan, Ann Arbor, Mich.
Wersn anu ie. 4 Se ane 24 Upper Mountain Ave., Montclair, N. J.
Weston, WitiiAM H., Jr., Ph.D., 716............ Fed. Hort. Board, Washington, D. C.
Viitewoporeio ty LO i eel eel Du COLO eee Gos ety ast her 79 Chapel St., Albany, N. Y.
i Pou oh B12 61 oh yt egg) Coed FAN 6 ck aa CR eee er er 2342 Albion Pl., St. Louis, Mo.
Waurtrnc, WittiamM J., ’15......U.S. Naval Gun Factory, Optical Shop, Rochester, N. Y.
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INDEX

A

Acanthocephala of North American
Birds 19

Agriculture, Genetics in Relation to, 268

Amebas, Three New Species of, 79

Anatomical Preparations, A Method of
Mounting, 58

Angle, Edward J., Development of the
Wolffian Body in Sus scrofa domesti-
cus, 215

Aquatic Microscopy for Beginners, 199

B

Biochemical Catalysts in Life and In-
dustry, 67

Birds, North American, Acanthocephala
of, 19

Body, Development of the Wolffian, in
Sus scrofa domesticus, 215

Branchiobdellid Worms from Michigan
Crawfishes, 49

C

Carriers of Disease, Insects as, 7

Cellulose, Nitrate, as a Substitute for
Calloidin, 269

Cestode Ova, Green Light for Demon-
strating, 59

Chickering, A. M.,
Ranatra, 132

Cort, William Walter, Methods for
Studying Living Trematodes, 129

Crawfishes, Michigan, Branchiobdellid
Worms from, 49

Custodian’s Report, 72

D
Diatom-Eating Flagellate, A New and
Remarkable, 177
Dickey, L. B., and Smith, F., A New
Species of Rynchelmis in North Ameri-
ca, 207

Chromosomes of

Disease, Insects as Carriers of, 7
Dog, Spermatogenesis of, 97

EB

Ellis, Max M., Branchiobdellid Worms
from Michigan Crawfishes, 49

Ellis, M. M., Green Light for Demon-
strating Living Cestode Ova, 59

Euparal, A Substitute for, 131

¥F

Faust, Ernest Carroll, Studies in Ameri-
can Stephanophialinae, 183

Faust, E. C., Additions to our Knowledge
of Unionicola Aculeata, 125

Flagellate, A New and Remarkable
Diatom-Eating, 177

Fresh Water Biology, 61

G
Genetics in Relation to Agriculture, 268
Green Light for Demonstrating Cestode
Ova, 59
H

Horizontal Distribution of Plankton,
Variation in the, 239
I
Insects as Carriers of Disease, 7
Introduction to the Study of Science, 64
1b
Latham, V. A., New Method of Staining
Tissues Containing Nerves, Fontana’s
Spirochete Stain, Simple Method of
Cleaning Old Slides, Menthol for
Marcotizing, 59
M
MacGregor, Malcolm Evan, Insects as
Carriers of Disease, 7

Malone, Julian Y., Spermatogenesis of
the Dog, 97

382

McCalla, Dr. Albert, 201

Menthol for Narcotizing, 60

Method of Mounting Anatomical Pre-
parations, 58

Microscopy, Aquatic, for Beginners, 199

Miller, Chas. H., Nitrate Cellulose as a
Substitute for Celloidin, 269

Minutes of Meeting, 71

Moberg, Eric, G., Variation in the Hori-
zontal Distribution of Plankton in
Devils Lake, North Dakota, 239

Mounting Anatomical Preparations, A
Method of, 58

N

Nitrate Cellulose as a Substitute for
Celloidin, 269
12
Parthenogenetic and Bisexual Nema-
todes, Reproduction in, 141
Plankton, Variation in the Horizontal
Distribution of, in Devils Lake,
North Dakota, 239
Plant Histology and Physiology, A
Chart on, 53
Pool, Raymond J., A Chart on General
Plant Histology and Physiology, 53
Preserving Marine Biological Specimens,
Methods of, 134
R

Reproduction in Parthenogenetic and
Bisexual Nematodes, 141
Rotifers, Notes on Collecting and
Mounting, 133
Rynchelmis, A New Species of, in North
America, 207
S

Schaeffer, Asa A., Three New Species
of Amebas, 79

Schaeffer, Asa A., A New and Remark-
able Diatom-Eating Flagelfate, 177

Scott, G. G., A Method of Mounting
Anatomical Preparations for Exhibi-
tion, 58

INDEX

Shepherd, E. S., A Substitute for Euparal,
131

Short History of Science, 65

Silvermann Illuminator for Microscopes,
136

Smith F., and Dickey, L. B., A New
Species of Rynchelmis in North
America, 207

Spermatogenesis of the Dog, 97

Spirochete Stain, Fontana’s, 60

Staining Tissues Containing Nerves, 59

Stephanophialinae, Studies in, 183

Sus scrofa domesticus, Development of
the Wolffian Body in, 215

Ak

Thigmotactic Reactions of Fresh Water
Turbellarian, Phagocata Gracilis, 111

. Three New Species of Amebas, 79

Treasurer’s Report, 73
Trematodes, Methods for Studying, 129

U

Unionicola Aculeata, Additions to our
Knowledge of, 125

V

Van Cleave, H. J., Acanthocephala of
North American Birds, 19

Variation in the Horizontal Distribution
of Plankton, 239

Ww

Wehrle, L. P., and Welch, Paul S.
Observations on Reproduction in
Nematodes, 141

Weimer, Bernol R., Thigmotactic Re-
actions of Fresh Water Turbellarian,
111

Welch, Paul S., and Wehrle, L. P., Ob-
servations on Reproduction in Nema-
todes, 141

Wolffian Body, Development of, in Sus
scrofa, domesticus, 215

Worms, Branchiobdellid, from Michigan
Crawfishes, 49

QH American Microscopical
201 Society

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